Human aminoacyl-tRNA synthetase polypeptides useful for the regulation of angiogenesis

ABSTRACT

Compositions comprising truncated tryptophanyl-tRNA synthetase polypeptides useful for regulating angiogenesis, as well as nucleic acids encoding such tRNA synthetase polypeptides are described. Methods of making and using such compositions are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 12/386,428,filed on Apr. 17, 2009, which is a continuation of U.S. patentapplication Ser. No. 11/444,924, filed on Jun. 1, 2006, now U.S. Pat.No. 7,521,215, which is a division of U.S. patent application Ser. No.10/240,532, filed on Sep. 30, 2002, now U.S. Pat. No. 7,067,126, whichis the National Stage of PCT/US01/08975, filed on Mar. 21, 2001, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/193,471,filed on Mar. 31, 2000, the disclosures of which are incorporated hereinby reference.

GOVERNMENTAL RIGHTS

This invention was made with governmental support from the United StatesGovernment, National Institutes of Health, Grant GM23562; the UnitedStates Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to compositions comprising truncated tRNAsynthetase polypeptides, as well as nucleic acids encoding suchtruncated tRNA synthetase polypeptides. Methods of making and using suchcompositions are also disclosed.

BACKGROUND OF THE INVENTION

Aminoacyl-tRNA synthetases, which catalyze the aminoacylation of tRNAmolecules, are ancient proteins that are essential for decoding geneticinformation during the process of translation. In higher eukaryotes,nine aminoacyl-tRNA synthetases associate with at least three otherpolypeptides to form a supramolecular multienzyme complex (Mirande etal., 1985, Eur. J. Biochem. 147:281-89). Each of the eukaryotic tRNAsynthetases consists of a core enzyme, which is closely related to theprokaryotic counterpart of the tRNA synthetase, and an additional domainthat is appended to the amino-terminal or carboxyl-terminal end of thecore enzyme (Mirande, 1991, Prog. Nucleic Acid Res. Mol. Biol.40:95-142). Human tyrosyl-tRNA synthetase (TyrRS), for example, has acarboxyl-terminal domain that is not part of prokaryotic and lowereukaryotic TyrRS molecules (FIG. 1) (Rho et al., 1998, J. Biol. Chem.273:11267-73). It has also been suggested that both the bovine andrabbit TyrRS molecules possess an extra domain (Kleeman et al., 1997, J.Biol. Chem. 272:14420-25).

In most cases, the appended domains appear to contribute to the assemblyof the multienzyme complex (Mirande, supra). However, the presence of anextra domain is not strictly correlated with the association of asynthetase into the multienzyme complex. Higher eukaryotic TyrRS, forexample, is not a component of the multienzyme complex (Mirande et al.,supra).

The carboxyl-terminal domain of human TyrRS shares a 51% sequenceidentity with the mature form of human endothelial monocyte-activatingpolypeptide II (EMAP II) (Rho et al., supra). TyrRS is the only highereukaryotic aminoacyl-tRNA synthetase known to contain an EMAP II-likedomain. The EMAP-like domain of TyrRS has been shown to be dispensablefor aminoacylation in vitro and in yeast (Wakasugi et al., 1998, EMBO J.17:297-305).

EMAP II is a proinflammatory cytokine that was initially identified as aproduct of murine methylcholanthrene A-induced fibrosarcoma cells.Pro-EMAP II is cleaved and is secreted from apoptotic cells to produce abiologically active 22-kD mature cytokine (Kao, et al., 1994, J. Biol.Chem. 269:25106-19). The mature EMAP II can induce migration ofmononuclear phagocytes (MPs) and polymorphonuclear leukocytes (PMNs); italso stimulates the production of tumor necrosis factor-α (TNF α) andtissue factor by MPs and the release of myeloperoxidase from PMNs.

The catalytic core domain of tryptophanyl-tRNA synthetase (TrpRS) is aclose homolog of the catalytic domain of TyrRS (Brown et al., 1997, J.Mol. Evol. 45:9-12). As shown in FIG. 15, mammalian TrpRS molecules havean amino-terminal appended domain. In normal human cells, two forms ofTrpRS can be detected: a major form consisting of the full-lengthmolecule and a minor truncated form (“mini TrpRS”). The minor form isgenerated by the deletion of the amino-terminal domain throughalternative splicing of the pre-mRNA (Tolstrup et al., 1995, J. Biol.Chem. 270:397-403). The amino-terminus of mini TrpRS has been determinedto be the met residue at position 48 of the full-length TrpRS molecule(id.). Alternatively, truncated TrpRS may be generated by proteolysis(Lemaire et al., 1975, Eur. J. Biochem. 51:237-52). For example, bovineTrpRS is highly expressed in the pancreas and is secreted into thepancreatic juice (Kisselev, 1993, Biochimie 75:1027-39), thus resultingin the production of a truncated TrpRS molecule. These results suggestthat truncated TrpRS has a function other than the aminoacylation oftRNA (Kisselev, supra).

Angiogenesis, or the proliferation of new capillaries from pre-existingblood vessels, is a fundamental process necessary for embryonicdevelopment, subsequent growth, and tissue repair. Angiogenesis is aprerequisite for the development and differentiation of the vasculartree, as well as for a wide variety of fundamental physiologicalprocesses including embryogenesis, somatic growth, tissue and organrepair and regeneration, cyclical growth of the corpus luteum andendometrium, and development and differentiation of the nervous system.In the female reproductive system, angiogenesis occurs in the follicleduring its development, in the corpus luteum following ovulation and inthe placenta to establish and maintain pregnancy. Angiogenesisadditionally occurs as part of the body's repair processes, e.g. in thehealing of wounds and fractures. Angiogenesis is also a factor in tumorgrowth, since a tumor must continuously stimulate growth of newcapillary blood vessels in order to grow. Angiogenesis is an essentialpart of the growth of human solid cancer, and abnormal angiogenesis isassociated with other diseases such as rheumatoid arthritis, psoriasis,and diabetic retinopathy (Folkman, J. and Klagsbrun, M., Science235:442-447, (1987)).

Several factors are involved in angiogenesis. Both acidic and basicfibroblast growth factor molecules that are mitogens for endothelialcells and other cell types. Angiotropin and angiogenin can induceangiogenesis, although their functions are unclear (Folkman, J., 1993,Cancer Medicine pp. 153-170, Lea and Febiger Press). A highly selectivemitogen for vascular endothelial cells is vascular endothelial growthfactor or VEGF (Ferrara, N., et al., Endocr. Rev. 13:19-32, (1992)).

SUMMARY OF THE INVENTION

The invention provides novel truncated tRNA synthetase polypeptideshaving chemokine activity that are useful for research, diagnostic,prognostic and therapeutic applications. In one embodiment, the tRNAsynthetase polypeptides are useful for regulating vascular endothelialcell function, and in particular, for regulating angiogenesis.

In one embodiment, the novel truncated tRNA synthetase polypeptidescomprise a Rossmann fold nucleotide binding domain wherein the isolatedpolypeptide is capable of regulating vascular endothelial cell function.In one embodiment, the truncated tRNA synthetase polypeptide is atyrosyl-tRNA synthetase with a carboxyl-terminal truncation. In anotherembodiment, the truncated tRNA synthetase polypeptide is atryptophanyl-tRNA synthetase, preferably at least about 46 kilodaltonsin size, with an amino-terminal truncation.

Both angiogenic and angiostatic truncated tRNA synthetase polypeptidesare provided by the present invention.

In one embodiment, preferred truncated tRNA synthetase polypeptidesinclude a polypeptide consisting essentially of amino acid residues1-364 of SEQ ID NO:2; a polypeptide consisting essentially of amino acidresidues 1-343 of SEQ ID NO:2; a polypeptide of approximately 40 kDmolecular weight produced by cleavage of the polypeptide of SEQ ID NO:2with polymorphonuclear leucocyte elastase; and fragments thereofcomprising the amino acid sequence -Glu-Leu-Arg-Val-Ser-Tyr-.

In another embodiment, preferred truncated tRNA synthetase polypeptidesinclude a polypeptide consisting essentially of amino acid residues48-471 of SEQ ID NO:10; a polypeptide consisting essentially of aminoacid residues 71-471 of SEQ ID NO:10; a polypeptide of approximately 47kD molecular weight produced by cleavage of the polypeptide of SEQ IDNO:10 with polymorphonuclear leucocyte elastase; and fragments thereofcomprising the amino acid sequence -Asp-Leu-Thr-. In one preferredembodiment, the truncated tRNA synthetase polypeptide is mammalian, andmore preferably, human.

In another embodiment, the invention comprises an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequence atleast 95% identical to a sequence selected from the group consisting ofa polynucleotide of SEQ ID NO:1 or SEQ ID NO: 9; a polynucleotide whichis hybridizable to a polynucleotide of SEQ ID NO:1 or SEQ ID NO: 9; apolynucleotide encoding the polypeptide of SEQ ID NO:2 or SEQ ID NO: 10;a polynucleotide encoding a polypeptide epitope of SEQ ID NO:2 or SEQ IDNO: 10 and a polynucleotide that is hybridizable to a polynucleotideencoding a polypeptide epitope of SEQ ID NO:2 or SEQ ID NO:10. In apreferred embodiment the invention comprises a recombinant expressionvector comprising the isolated nucleic acid molecule of encoding a tRNAsynthetase polypeptide. Another embodiment is a host cell comprising arecombinant expression vector comprising the isolated nucleic acidmolecule of SEQ ID NO:1 or SEQ ID NO:9 encoding a tRNA synthetasepolypeptide.

In one embodiment, the present invention is a process for making tRNAsynthetase polypeptides by treating tyrosyl-tRNA synthetase with aprotease. In one embodiment, the present invention is a process formaking tRNA synthetase polypeptides by treating tryptophanyl-tRNAsynthetase with a protease. One preferred protease is polymorphonuclearleukocyte elastase.

The invention provides compositions comprising tRNA synthetasepolypeptides and a pharmaceutically suitable excipient. Suchcompositions are suitable for transdermal, transmucosal, enteral orparenteral administration. In another embodiment, the tRNA synthetasepolypeptide can be used for the preparation of a pharmaceuticalcomposition for transdermal, transmucosal, enteral or parenteraladministration.

In one embodiment, the tRNA synthetase polypeptide can have angiogenicactivity at least two-fold greater than control levels. In embodimentsin which the tRNA synthetase polypeptide has angiostatic activity, thepolypeptide suppresses at least ten percent of angiogenic activity, morepreferably at least ninety percent of angiogenic activity.

The invention further provides a method of enhancing angiogenesis in amammal comprising the step of administering an angiogenically effectiveamount of a composition comprising a angiogenic tRNA synthetasepolypeptide and a pharmaceutically suitable excipient.

The invention further provides a method of suppressing angiogenesis in amammal comprising the step of administering an angiostatically effectiveamount of a composition comprising an angiostatic tRNA synthetasepolypeptide and a pharmaceutically suitable excipient.

In another embodiment, the invention provides a method of enhancingangiogenesis to a graft in a mammal comprising the step of administeringan angiogenically effective amount of a composition comprising anangiogenic tRNA synthetase polypeptide and a pharmaceutically suitableexcipient.

In another embodiment, the invention provides a method of treatingmyocardial infarction in a mammal comprising the step of administeringan angiogenically effective amount of a composition comprising anangiogenic tRNA synthetase polypeptide and a pharmaceutically suitableexcipient.

In another embodiment, the invention provides a method of treating acondition that would benefit from increased angiogenesis in a mammalcomprising the step of administering an angiogenically effective amountof a composition comprising an angiogenic tRNA synthetase polypeptideand a pharmaceutically suitable excipient.

In another embodiment, the invention provides a method of treating acondition that would benefit from decreased angiogenesis in a mammalcomprising the step of administering an angiostatically effective amountof the composition comprising an angiostatic tRNA synthetase polypeptideand a pharmaceutically suitable excipient.

In another embodiment, the invention provides a method of treating asolid tumor in a mammal comprising the step of administering anangiostatically effective amount of the composition comprising anangiostatic tRNA synthetase polypeptide and a pharmaceutically suitableexcipient.

In another embodiment, the invention provides a method of suppressingtumor metatasis in a mammal comprising the step of administering anangiostatically effective amount of the composition comprising anangiostatic tRNA synthetase polypeptide and a pharmaceutically suitableexcipient.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic alignment of E. coli TyrRS, humanfull-length TyrRS, human mini TyrRS, the human TyrRS carboxyl-terminaldomain, and human mature EMAP II;

FIG. 2 illustrates the effects of human full-length TyrRS, human miniTyrRS, the human TyrRS carboxyl-terminal domain, human mature EMAP II,and E. coli TyrRS on MP chemotaxis (white bars) and MP TNFμ production(gray bars);

FIG. 3 illustrates the effects of human full-length TyrRS, human miniTyrRS, the human TyrRS carboxyl-terminal domain, human mature EMAP II,and E. coli TyrRS on MP tissue factor production (white bars) and PMNrelease of myeloperoxidase (gray bars);

FIG. 4 illustrates the effects of human full-length TyrRS, human miniTyrRS, the human TyrRS carboxyl-terminal domain, human mature EMAP II,E. coli TyrRS, human TyrRS mutant, and IL-8 on PMN chemotaxis;

FIG. 5 illustrates a schematic partial alignment of human mini TyrRS(SEQ ID NO: 17), E. coli TyrRS (SEQ ID NO: 18), IL-8 (SEQ ID NO: 19),Groα (SEQ ID NO: 20), and NAP-2 (SEQ ID NO: 21); the connectivepolypeptide 1 (CP1) which splits the Rossman nucleotide-binding fold inTyrRS is indicated;

FIG. 6 illustrates the results of competition assays in which unlabeledhuman mini TyrRS, human TyrRS mutant, human full-length TyrRS, the humanTyrRS carboxyl-terminal domain, human mature EMAP II, E. coli TyrRS,IL-8, Groα, or NAP-2 was used in molar excess with ¹²⁵I-human mini TyrRSon PMNs;

FIGS. 7A-7B illustrate the results of immunoblot analysis of (A)supernatants following growth of U-937 cells in normal media (lane 1)and serum-free media (lane 2), and (B) supernatants following growth ofU-937 cells in serum-free media for 4, 12, or 24 hours (lanes 1-3), orfrom a cell extract isolated from U-937 cells grown in serum-free mediafor 24 hours;

FIGS. 8A-8B illustrate (A) the results of immunoblot analysis of humanfull-length TyrRS (lane 1), human mini TyrRS (lane 2), the TyrRScarboxyl-terminal domain (lane 3), the extended carboxyl-terminal domainof human TyrRS (lane 4), and human full-length TyrRS following cleavagewith PMN elastase (lane 5), and (B) a schematic representation of thecleavage sites for human pro-EMAP II (SEQ ID NO: 22) and humanfull-length TyrRS (SEQ ID NO: 23);

FIG. 9 illustrates a schematic alignment of human full-length TyrRS,human mini TyrRS, S. cerevisiae TyrRS, and E. coli TyrRS;

FIG. 10 illustrates the results of competition assays in which unlabeledhuman mini TyrRS, S. cerevisiae TyrRS, or E. coli TyrRS was used inmolar excess with ¹²⁵I-human mini TyrRS on PMNs;

FIG. 11 illustrates the effects of human mini TyrRS, S. cerevisiaeTyrRS, or E. coli TyrRS on PMN chemotaxis;

FIG. 12 illustrates a schematic alignment of the human EMAP II-likedomains and corresponding synthetic peptides for human full-length TyrRS(SEQ ID NO: 24), human EMAP II (SEQ ID NO: 25-26), C. elegans MetRS (SEQID NO: 27), and S. cervisiae Arc1p (SEQ ID NO: 28);

FIG. 13 illustrates the effects of synthetic peptides derived from humanTyrRS, human EMAP II, C. elegans MetRS, and S. cervisiae Arc1p on PMNchemotaxis

FIG. 14 illustrates a schematic comparison between human mini TyrRS andα-chemokines (the carboxyl-terminal portions of each have been omitted);the location of β-sheets (solid arrows) and ELR motifs (circles) areindicated;

FIG. 15 illustrates a schematic alignment of human full-length TyrRS,human mini TyrRS, human full-length TrpRS, and human mini TrpRS; thecarboxyl-terminal and amino-terminal appended domains (hatched) areindicated;

FIG. 16 illustrates the angiogenic activity of human full-length TyrRS,human mini TyrRS, human mini TyrRS mutant, or human VEGF on HUVECchemotaxis;

FIG. 17 illustrates the angiogenic activity of human full-length TyrRS,human mini TyrRS, human mini TyrRS mutant, human mini TyrRS+IP-10, humanVEGF, or human VEGF+IP-10 on the CAMs of 10-day-old chick embryos;

FIG. 18 illustrates the angiogenic activity of human full-length TyrRSor human mini TyrRS on endothelial cell proliferation;

FIG. 19 illustrates the effect of human full-length TrpRS or human miniTrpRS on human VEGF-induced or human mini TyrRS-induced endothelialmigration;

FIG. 20 illustrates the effect of human full-length TrpRS or human miniTrpRS on the angiogenic activity of human VEGF or human mini TyrRS onchick CAM;

FIG. 21 illustrates the results of immunoblot analysis showing thesecretion of human TrpRS from human U-937 cells;

FIG. 22 illustrates the cleavage of human full-length TrpRS by PMNelastase; TrpRS was exposed to PMN elastase for 0 minutes (lane 1), 15minutes (lane 2), 30 minutes (lane 3), or 60 minutes (lane 4);

FIG. 23 illustrates the effect of human supermini TrpRS on theangiogenic activity of human VEGF or human mini TyrRS on chick CAM;

FIG. 24 illustrates the amino acid sequence similarity of human TrpRS(SEQ ID NO: 45 and SEQ ID NO: 51), bovine TrpRS (SEQ ID NO: 46 and SEQID NO: 52), mouse TrpRS (SEQ ID NO: 47 and SEQ ID NO: 53), rabbit TrpRS(SEQ ID NO: 48 and SEQ ID NO: 54), human semaphorin-E (SEQ ID NO: 49),mouse semaphorin-E (SEQ ID NO: 50), and mouse neuropilin-2 (SEQ ID NO:55); the amino-terminal residue of the mini, supermini, and inactiveforms of TrpRS (arrows), identical residues (asterisks), semi-conservedresidues (dots), and insertions in the c-domain of neurophilin-2 (bars)are indicated;

FIG. 25 illustrates the regions of semaphorin-E and neuropilin-2 thatshare sequence similarity with mammalian TrpRS (indicated by arrows);semaphorin-E and neuropilin-2 domains are indicated as follows:semaphorin domain (sema), immunoglobulin domain (Ig), carboxy-terminalbasic domain (C), complement-binding domain (a1, a2), coagulation factordomain (b1, b2), c domain (c), transmembrane domain (TM), andcytoplasmic domain (Cy). Isoforms of neuropilin exist with insertions of5, 17, or 22 amino acids in the c-domain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In order to facilitate understanding of the following examples, certainfrequently occurring methods and/or terms will be described.

The term “tRNA synthetase polypeptides” means polypeptides that areshorter than the corresponding full length tRNA synthetase.

As used herein the term “cell culture” encompasses both the culturemedium and the cultured cells. As used herein the phrase “isolating apolypeptide from the cell culture” encompasses isolating a soluble orsecreted polypeptide from the culture medium as well as isolating anintegral membrane protein from the cultured cells.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μL of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a poly-acrylamide gel to isolate the desired fragment.“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

As used herein “angiogenic TyrRS peptides” refers to fragments of TyrRShaving angiogenic activity, including, but not limited to, mini TyrRS,fragments, analogs and derivatives thereof comprising the amino acidsequence ELR.

As used herein, “TyrRS angiogenic therapy” refers to the delivery of anangiogenic effective amount of mini TyrRS or fragments thereof as wellas to the delivery of polynucleotides encoding an angiogenic effectiveamount of mini TyrRS or fragments thereof.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in SEQ ID NO:1 or in SEQ ID NO:9 or may be adifferent coding sequence which coding sequence, as a result of theredundancy or degeneracy of the genetic code, encodes the same, maturepolypeptide sequence shown in SEQ ID NO:2 or in SEQ ID NO:10.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence. The present invention further relates to variantsof the hereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:10 or the polypeptide encoded bythe cDNA of SEQ ID NO:1 or SEQ ID NO:9. The variant of thepolynucleotide may be a naturally occurring allelic variant of thepolynucleotide or a non-naturally occurring variant of thepolynucleotide. Thus, the present invention includes polynucleotidesencoding the same mature polypeptide as shown of SEQ ID NO:2 or SEQ IDNO:10 or the same mature polypeptide encoded by the cDNA of SEQ ID NO:1or SEQ ID NO:9 as well as variants of such polynucleotides whichvariants encode for an fragment, derivative or analog of the polypeptideof SEQ ID NO:2 or SEQ ID NO:10 or the polypeptide encoded by SEQ ID NO:1or SEQ ID NO:9. Such nucleotide variants include deletion variants,substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in SEQ ID NO:1 or SEQ ID NO:9. As known in the art, an allelicvariant is an alternate form of a polynucleotide sequence which have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide which aids in expression and secretion of apolypeptide from a host cell, for example, a leader sequence whichfunctions as a secretory sequence for controlling transport of apolypeptide from the cell. The polypeptide having a leader sequence is apreprotein and may have the leader sequence cleaved by the host cell toform the mature form of the polypeptide. The polynucleotides may alsoencode for a proprotein which is the mature protein plus additional 5′amino acid residues. A mature protein having a prosequence is aproprotein and is an inactive form of the protein. Once the prosequenceis cleaved an active mature protein remains. Thus, for example, thepolynucleotide of the present invention may encode for a mature protein,or for a protein having a prosequence or for a protein having both aprosequence and presequence (leader sequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 50% andpreferably 70% identity between the sequences. The present inventionparticularly relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides. As herein used,the term “stringent conditions” means hybridization will occur only ifthere is at least 95% and preferably at least 97% identity between thesequences. The polynucleotides which hybridize to the hereinabovedescribed polynucleotides in a preferred embodiment encode polypeptideswhich retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNA of SEQ ID NO:1 or SEQ IDNO:9.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide or that encoded by the deposited cDNA, means a polypeptidewhich retains essentially the same biological function or activity assuch polypeptide. Thus, an analog includes a proprotein which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of SEQ ID NO:2 orSEQ ID NO:10 or that encoded by the polynucleotide of SEQ ID NO:1 or SEQID NO:9 may be (I) one in which one or more of the amino acid residuesare substituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the tRNA synthetase polpeptide genes. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotide of the present invention may be employed forproducing a polypeptide by recombinant techniques. Thus, for example,the polynucleotide sequence may be included in any one of a variety ofexpression vehicles, in particular vectors or plasmids for expressing apolypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus,and pseudorabies. A preferred vector is pET20b. However, any otherplasmid or vector may be used as long as it is replicable and viable inthe host.

As hereinabove described, the appropriate DNA sequence may be insertedinto the vector by a variety of procedures. In general, the DNA sequenceis inserted into an appropriate restriction endonuclease sites byprocedures known in the art. Such procedures and others are deemed to bewithin the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain a gene to providea phenotypic trait for selection of transformed host cells such asdihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as herein abovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Salmonellatyphimurium, Streptomyces; fungal cells, such as yeast; insect cells,such as Drosophila and Sf9; animal cells such as CHO, COS or Bowesmelanoma; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE-9 (Qiagen), pBs,phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a,pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, PRIT5(Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, PMSG, pSVL (Pharmacia) and pET20B. In one preferredembodiment, the vector is pET20B. However, any other plasmid or vectormay be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), PLand Trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described construct. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, 1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook. et al., Molecular Cloning: ALaboratory Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989), thedisclosure of which is hereby incorporated by reference. Transcriptionof a DNA encoding the polypeptides of the present invention by highereukaryotes is increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100 to 270), a cytomegalovirus early promoter enhancer, apolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification. Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 viralgenome, for example, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

Polypeptides are recovered and purified from recombinant cell culturesby methods used heretofore, including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxyapatite chromatography and lectinchromatography. It is preferred to have low concentrations(approximately 0.1-5 mM) of calcium ion present during purification(Price, et al., J. Biol. Chem., 244:917 (1969)). Protein refolding stepscan be used, as necessary, in completing configuration of the matureprotein. Finally, high performance liquid chromatography (HPLC) can beemployed for final purification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated with mammalian or other eukaryotic carbohydrates or may benon-glycosylated.

The polypeptides of the present invention may be modified to improvestability and increase potency by means known in the art. For example,L-amino acids can be replaced by D-amino acids, the amino terminus canbe acetylated, or the carboxyl terminus modified, e.g.,ethylamine-capped (Dawson, D. W., et al., Mol. Pharmacol., 55: 332-338(1999)).

Angiogenic tRNA synthetase polypeptides are useful as wound healingagents, particularly where it is necessary to re-vascularize damagedtissues, or where new capillary angiogenesis is important. Therefore, itmay be used for treatment of full-thickness wounds such as dermalulcers, including pressure sores, venous ulcers, and diabetic ulcers. Inaddition, it can be used in the treatment of full-thickness burns andinjuries where angiogenesis is desired to prepare the burn in injuredsites for a skin graft and flap. In this case, it should be applieddirectly at the sites. Similarly angiogenic tRNA synthetase polypeptidespolypeptides can be used in plastic surgery when reconstruction isrequired following a burn, other trauma, or even for cosmetic purposes.

Since angiogenesis is important in keeping wounds clean andnon-infected, angiogenic tRNA synthetase polypeptides may be used inassociation with surgery and following the repair of cuts. It should beparticularly useful in the treatment of abdominal wounds where there isa high risk of infection.

Angiogenic tRNA synthetase polypeptides can be used for the promotion ofendothelialization in vascular graft surgery. In the case of vasculargrafts using either transplanted or synthetic material, angiogenic TyrRSpeptides can be applied to the surface of the graft, preferably in apharmaceutically appropriate excipient. In one embodiment, thepharmaceutically appropriate excipient further provides for thecontinuous release of angiogenic TyrRS peptides.

Angiogenic tRNA synthetase therapy can be used to repair the damage ofmyocardial infarction. In one preferred embodiment, angiogenic TyrRStherapy can be used in conjunction with coronary bypass surgery bystimulating the growth of the transplanted tissue. In one preferredembodiment, angiogenic tRNA synthetase therapy can be administered bydirect myocardial injection of angiogenic tRNA synthetase polypeptidesor polynucleotides encoding angiogenic tRNA synthetase polypeptides. SeeLosodo, D. W., et al., Circulation, 98: 2800-2804 (1998).

In another embodiment, angiogenic tRNA synthetase therapy can be used inconjunction with angiography to administer the angiogenic tRNAsynthetase polypeptides or polynucleotides encoding angiogenic tRNAsynthetase polypeptides directly to the lumen and wall of the bloodvessel.

Similarly, tRNA synthetase therapy can be used to administer theangiogenic or angiostatic tRNA synthetase polypeptides orpolynucleotides encoding angiogenic or angiostatic tRNA synthetasepolypeptides directly to the lumen and wall of other hollow organs, suchas the uterus.

The polypeptide of the present invention may also be employed inaccordance with the present invention by expression of such polypeptidein vivo, which is often referred to as “gene therapy.”

Thus, for example, cells such as bone marrow cells may be engineeredwith a polynucleotide (DNA or RNA) encoding for the polypeptide ex vivo,the engineered cells are then provided to a patient to be treated withthe polypeptide. Such methods are well-known in the art. For example,cells may be engineered by procedures known in the art by use of aretroviral particle containing RNA encoding for the polypeptide of thepresent invention. Similarly, cells may be engineered in vivo forexpression of the polypeptide in vivo, for example, by procedures knownin the art. As known in the art, a producer cell for producing aretroviral particle containing RNA encoding the polypeptide of thepresent invention may be administered to a patient for engineering cellsin vivo and expression of the polypeptide in vivo. These and othermethods for administering a polypeptide of the present invention by suchmethods should be apparent to those skilled in the art from theteachings of the present invention. For example, the expression vehiclefor engineering cells may be other than a retroviral particle, forexample, an adenovirus, which may be used to engineering cells in vivoafter combination with a suitable delivery vehicle.

Various viral vectors that can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, adeno-associatedvirus (AAV), or, preferably, an RNA virus such as a retrovirus.Preferably, the retroviral vector is a derivative of a murine or avianretrovirus, or is a lentiviral vector. The preferred retroviral vectoris a lentiviral vector. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. By inserting a zinc finger derived-DNA binding polypeptidesequence of interest into the viral vector, along with another gene thatencodes the ligand for a receptor on a specific target cell, forexample, the vector is made target specific. Retroviral vectors can bemade target specific by inserting, for example, a polynucleotideencoding a protein. Preferred targeting is accomplished by using anantibody to target the retroviral vector. Those of skill in the art willknow of, or can readily ascertain without undue experimentation,specific polynucleotide sequences which can be inserted into theretroviral genome to allow target specific delivery of the retroviralvector containing the zinc finger-nucleotide binding proteinpolynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include but are not limited to ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced. The vector virions produced by thismethod can then be used to infect a tissue cell line, such as NIH 3T3cells, to produce large quantities of chimeric retroviral virions.

Another targeted delivery system for polynucleotides encoding zincfinger derived-DNA binding polypeptides is a colloidal dispersionsystem. Colloidal dispersion systems include macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Thepreferred colloidal system of this invention is a liposome. Liposomesare artificial membrane vesicles which are useful as delivery vehiclesin vitro and in vivo. It has been shown that large unilamellar vesicles(LUV), which range in size from 0.2-4.0 μm can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand be delivered to cells in a biologically active form (Fraley, et al.,Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells,liposomes have been used for delivery of polynucleotides in plant, yeastand bacterial cells. In order for a liposome to be an efficient genetransfer vehicle, the following characteristics should be present: (1)encapsulation of the genes of interest at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes has been classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

In general, the compounds bound to the surface of the targeted deliverysystem will be ligands and receptors which will allow the targeteddelivery system to find and “home in” on the desired cells. A ligand maybe any compound of interest which will bind to another compound, such asa receptor.

In general, surface membrane proteins which bind to specific effectormolecules are referred to as receptors. In the present invention,antibodies are preferred receptors. Antibodies can be used to targetliposomes to specific cell-surface ligands. For example, certainantigens expressed specifically on tumor cells, referred to astumor-associated antigens (TAAs), may be exploited for the purpose oftargeting antibody-zinc finger-nucleotide binding protein-containingliposomes directly to the malignant tumor. Since the zincfinger-nucleotide binding protein gene product may be indiscriminatewith respect to cell type in its action, a targeted delivery systemoffers a significant improvement over randomly injecting non-specificliposomes. A number of procedures can be used to covalently attacheither polyclonal or monoclonal antibodies to a liposome bilayer.Antibody-targeted liposomes can include monoclonal or polyclonalantibodies or fragments thereof such as Fab, or F(ab′)₂, as long as theybind efficiently to an the antigenic epitope on the target cells.Liposomes may also be targeted to cells expressing receptors forhormones or other serum factors.

There are available to one skilled in the art multiple viral andnon-viral methods suitable for introduction of a nucleic acid moleculeinto a target cell. Genetic manipulation of primary tumor cells has beendescribed previously (Patel et al., 1994). Genetic modification of acell may be accomplished using one or more techniques well known in thegene therapy field (Human Gene Therapy, April 1994, Vol. 5, p. 543-563;Mulligan, R. C. 1993). Viral transduction methods may comprise the useof a recombinant DNA or an RNA virus comprising a nucleic acid sequencethat drives or inhibits expression of a protein having sialyltransferaseactivity to infect a target cell. A suitable DNA virus for use in thepresent invention includes but is not limited to an adenovirus (Ad),adeno-associated virus (AAV), herpes virus, vaccinia virus or a poliovirus. A suitable RNA virus for use in the present invention includesbut is not limited to a retrovirus or Sindbis virus. It is to beunderstood by those skilled in the art that several such DNA and RNAviruses exist that may be suitable for use in the present invention.

Adenoviral vectors have proven especially useful for gene transfer intoeukaryotic cells (Stratford-Perricaudet and Perricaudet. 1991).Adenoviral vectors have been successfully utilized to study eukaryoticgene expression (Levrero, M., et al. 1991). vaccine development (Grahamand Prevec, 1992), and in animal models (Stratford-Perricaudet, et al.1992.; Rich, et al. 1993). The first trial of Ad-mediated gene therapyin human was the transfer of the cystic fibrosis transmembraneconductance regulator (CFTR) gene to lung (Crystal, et al., 1994).Experimental routes for administrating recombinant Ad to differenttissues in vivo have included intratracheal instillation (Rosenfeld, etal. 1992) injection into muscle (Quantin, B., et al. 1992), peripheralintravenous injection (Herz and Gerard, 1993) and stereotacticinoculation to brain (Le Gal La Salle, et al. 1993). The adenoviralvector, then, is widely available to one skilled in the art and issuitable for use in the present invention.

Adeno-associated virus (AAV) has recently been introduced as a genetransfer system with potential applications in gene therapy. Wild-typeAAV demonstrates high-level infectivity, broad host range andspecificity in integrating into the host cell genome (Hermonat andMuzyczka. 1984). Herpes simplex virus type-1 (HSV-1) is attractive as avector system, especially for use in the nervous system because of itsneurotropic property (Geller and Federoff. 1991; Glorioso, et al. 1995).Vaccinia virus, of the poxvirus family, has also been developed as anexpression vector (Smith and Moss, 1983; Moss, 1992). Each of theabove-described vectors are widely available to one skilled in the artand would be suitable for use in the present invention.

Retroviral vectors are capable of infecting a large percentage of thetarget cells and integrating into the cell genome (Miller and Rosman.1989). Retroviruses were developed as gene transfer vectors relativelyearlier than other viruses, and were first used successfully for genemarking and transducing the cDNA of adenosine deaminase (ADA) into humanlymphocytes. Preferred retroviruses include lentiviruses. In preferredembodiments, the retrovirus is selected from the group consisting ofHIV, BIV and SIV.

“Non-viral” delivery techniques that have been used or proposed for genetherapy include DNA-ligand complexes, adenovirus-ligand-DNA complexes,direct injection of DNA, CaPO₄ precipitation, gene gun techniques,electroporation, liposomes and lipofection (Mulligan, 1993). Any ofthese methods are widely available to one skilled in the art and wouldbe suitable for use in the present invention. Other suitable methods areavailable to one skilled in the art, and it is to be understood that thepresent invention may be accomplished using any of the available methodsof transfection. Several such methodologies have been utilized by thoseskilled in the art with varying success (Mulligan, R. 1993). Lipofectionmay be accomplished by encapsulating an isolated DNA molecule within aliposomal particle and contacting the liposomal particle with the cellmembrane of the target cell. Liposomes are self-assembling, colloidalparticles in which a lipid bilayer, composed of amphiphilic moleculessuch as phosphatidyl serine or phosphatidyl choline, encapsulates aportion of the surrounding media such that the lipid bilayer surrounds ahydrophilic interior. Unilammellar or multilammellar liposomes can beconstructed such that the interior contains a desired chemical, drug,or, as in the instant invention, an isolated DNA molecule.

The cells may be transfected in vivo, ex vivo, or in vitro. The cellsmay be transfected as primary cells isolated from a patient or a cellline derived from primary cells, and are not necessarily autologous tothe patient to whom the cells are ultimately administered. Following exvivo or in vitro transfection, the cells may be implanted into a host.Genetic manipulation of primary tumor cells has been describedpreviously (Patel et al. 1994). Genetic modification of the cells may beaccomplished using one or more techniques well known in the gene therapyfield (Human Gene Therapy. April 1994. Vol. 5, p. 543-563; Mulligan, R.C. 1993).

In order to obtain transcription of the nucleic acid of the presentinvention within a target cell, a transcriptional regulatory regioncapable of driving gene expression in the target cell is utilized. Thetranscriptional regulatory region may comprise a promoter, enhancer,silencer or repressor element and is functionally associated with anucleic acid of the present invention. Preferably, the transcriptionalregulatory region drives high level gene expression in the target cell.Transcriptional regulatory regions suitable for use in the presentinvention include but are not limited to the human cytomegalovirus (CMV)immediate-early enhancer/promoter, the SV40 early enhancer/promoter, theJC polyomavirus promoter, the albumin promoter, PGK and the α-actinpromoter coupled to the CMV enhancer (Doll, et at 1996).

The vectors of the present invention may be constructed using standardrecombinant techniques widely available to one skilled in the art. Suchtechniques may be found in common molecular biology references such asMolecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), and PCR Protocols: A Guide to Methods and Applications(Innis, et al. 1990. Academic Press, San Diego, Calif.).

Administration of a nucleic acid of the present invention to a targetcell in vivo may be accomplished using any of a variety of techniqueswell known to those skilled in the art.

The vectors of the present invention may be administered orally,parentally, by inhalation spray, rectally, or topically in dosage unitformulations containing conventional pharmaceutically acceptablecarriers, adjuvants, and vehicles. The term parenteral as used hereinincludes, subcutaneous, intravenous, intramuscular, intrasternal,infusion techniques or intraperitoneally. Suppositories for rectaladministration of the drug can be prepared by mixing the drug with asuitable non-irritating excipient such as cocoa butter and polyethyleneglycols that are solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum and release the drug.

The dosage regimen for treating a disorder or a disease with the vectorsof this invention and/or compositions of this invention is based on avariety of factors, including the type of disease, the age, weight, sex,medical condition of the patient, the severity of the condition, theroute of administration, and the particular compound employed. Thus, thedosage regimen may vary widely, but can be determined routinely usingstandard methods.

The pharmaceutically active compounds (i.e., vectors) of this inventioncan be processed in accordance with conventional methods of pharmacy toproduce medicinal agents for administration to patients, includinghumans and other mammals. For oral administration, the pharmaceuticalcomposition may be in the form of, for example, a capsule, a tablet, asuspension, or liquid. The pharmaceutical composition is preferably madein the form of a dosage unit containing a given amount of DNA or viralvector particles (collectively referred to as “vector”). For example,these may contain an amount of vector from about 10³-10¹⁵ viralparticles, preferably from about 10⁶-10¹² viral particles. A suitabledaily dose for a human or other mammal may vary widely depending on thecondition of the patient and other factors, but, once again, can bedetermined using routine methods. The vector may also be administered byinjection as a composition with suitable carriers including saline,dextrose, or water.

While the nucleic acids and/or vectors of the invention can beadministered as the sole active pharmaceutical agent, they can also beused in combination with one or more vectors of the invention or otheragents. When administered as a combination, the therapeutic agents canbe formulated as separate compositions that are given at the same timeor different times, or the therapeutic agents can be given as a singlecomposition.

The polypeptide of the present invention may be employed in combinationwith a suitable pharmaceutical carrier. Such compositions comprise atherapeutically effective amount of the protein, and a pharmaceuticallyacceptable carrier or excipient. Such a carrier includes but is notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The formulation should suit the mode ofadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptide of the present invention may be employed on conjunction withother therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner, such as the transdermal, transmucosal, enteral and parenteralintravenous routes. The amounts and dosage regimens of tRNA synthetasepolypeptides administered to a subject will depend on a number offactors, such as the mode of administration, the nature of the conditionbeing treated, the body weight of the subject being treated and thejudgment of the prescribing physician. Generally speaking, it is given,for example, in therapeutically effective doses of at least about 10μg/kg body weight and, in most cases, it would be administered in anamount not in excess of about 8 mg/kg body weight per day and preferablythe dosage is from about 10 μg/kg body weight to about 1 μg/kg bodyweight daily, taking into the account the routes of administration,symptoms, etc.

The present invention is further directed to inhibiting tRNA synthetasepolypeptides in vivo by the use of antisense technology. Antisensetechnology can be used to control gene expression through triple-helixformation or antisense DNA or RNA, both of which methods are based onbinding of a polynucleotide to DNA or RNA. For example, the 5′ codingportion of the mature polynucleotide sequence, which encodes for thepolypeptide of the present invention, is used to design an antisense RNAoligonucleotide of from 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervanet al., Science, 251: 1360 (1991), thereby preventing transcription andthe production of VEGF2. The antisense RNA oligonucleotide hybridizes tothe mRNA in vivo and blocks translation of an mRNA molecule into thetRNA synthetase polypeptides (Okano, J. Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)).

Alternatively, the oligonucleotides described above can be delivered tocells by procedures in the art such that the anti-sense RNA or DNA maybe expressed in vivo to inhibit production of tRNA synthetasepolypeptides in the manner described above. Antisense constructs to tRNAsynthetase polypeptides, therefore, may inhibit the activity of the tRNAsynthetase polypeptides and prevent the further growth or even regresssolid tumors, since angiogenesis and neovascularization are essentialsteps in solid tumor growth. These antisense constructs may also be usedto treat rheumatoid arthritis, psoriasis and diabetic retinopathy whichare all characterized by abnormal angiogenesis.

Alternatively, angiostatic TrpRS therapy can be used to oppose theoppose the angiogenic activity of endogenous and exogenous angiogenicfactors, including TyrRS polypeptides, and to prevent the further growthor even regress solid tumors, since angiogenesis and neovascularizationare essential steps in solid tumor growth. Such therapies can also beused to treat rheumatoid arthritis, psoriasis and diabetic retinopathywhich are all characterized by abnormal angiogenesis.

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptide corresponding to a sequenceof the present invention can be obtained by direct injection of thepolypeptide into an animal or by administering the polypeptide to ananimal, preferably a nonhuman. The antibody so obtained will then bindthe polypeptide itself. In this manner, even a sequence encoding only afragment of the polypeptide can be used to generate antibodies bindingthe whole native polypeptide. Such antibodies can then be used toisolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention.

Neutralization antibodies can be identified and applied to mask theactivity of tRNA synthetase polypeptides. The utility of such anapproach has been shown in mice model systems against VEGF. VEGF2 canalso be inactivated by certain dominant negative mutants within the geneitself. It is known that both PDGF α and β form either heterodimers orhomodimers, and VEGF forms homodimers. These antibodies therefore may beused to block endogenous angiogenic activity and retard the growth ofsolid tumors. These antibodies may also be used to treat inflammationcaused by the increased vascular permeability. These antibodies mayfurther be used in an immunoassay to detect the presence of tumors incertain individuals. Enzyme immunoassay can be performed from the bloodsample of an individual.

The present invention is also directed to antagonist/inhibitors of thepolypeptides of the present invention. The antagonist/inhibitors arethose which inhibit or eliminate the function of the polypeptide. Thus,for example, antagonists bind to a polypeptide of the present inventionand inhibit or eliminate its function. The antagonist, for example,could be an antibody against the polypeptide which binds to thepolypeptide or, in some cases, an oligonucleotide. An example of aninhibitor is a small molecule which binds to and occupies the catalyticsite of the polypeptide thereby making the catalytic site inaccessibleto substrate such that normal biological activity is prevented. Examplesof small molecules include but are not limited to small peptides orpeptide-like molecules.

Alternatively, antagonists to the polypeptides of the present inventionmay be employed which bind to the receptors to which a polypeptide ofthe present invention normally binds. The antagonists may be closelyrelated proteins such that they recognize and bind to the receptor sitesof the natural protein, however, they are inactive forms of the naturalprotein and thereby prevent the action of the normal polypeptide ligand.The antagonist/inhibitors may be used therapeutically as an anti-tumordrug by occupying the receptor sites of tumors. Theantagonist/inhibitors may also be used to prevent inflammation. Theantagonist/inhibitors may also be used to treat solid tumor growth,diabetic retinopathy, psoriasis and rheumatoid arthritis. Theantagonist/inhibitors may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinabove described.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

Also preferred is a method for diagnosing in a subject a pathologicalcondition associated with abnormal structure or expression of a gene,which method comprises a step of detecting in a biological sampleobtained from said subject nucleic acid molecules, if any, comprising anucleotide sequence that is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from the groupconsisting of a nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:9.

The method for diagnosing a pathological condition can comprise a stepof detecting nucleic acid molecules comprising a nucleotide sequence ina panel of at least two nucleotide sequences, wherein at least onesequence in said panel is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a composition of matter comprising isolated nucleicacid molecules wherein the nucleotide sequences of said nucleic acidmolecules comprise a panel of at least two nucleotide sequences, whereinat least one sequence in said panel is at least 95% identical to asequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:1 andSEQ ID NO:9. The nucleic acid molecules can comprise DNA molecules orRNA molecules.

Further preferred is a method for detecting in a biological sample apolypeptide comprising an amino acid sequence which is at least 90%identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of amino acid sequences ofSEQ ID NO:2 and SEQ ID NO:10, which method comprises a step of comparingan amino acid sequence of at least one polypeptide molecule in saidsample with a sequence selected from said group and determining whetherthe sequence of said polypeptide molecule in said sample is at least 90%identical to said sequence of at least 10 contiguous amino acids.

Also preferred is the above method for identifying the species, tissueor cell type of a biological sample, which method comprises a step ofdetecting polypeptide molecules comprising an amino acid sequence in apanel of at least two amino acid sequences, wherein at least onesequence in said panel is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the abovegroup.

Also preferred is a method for diagnosing in a subject a pathologicalcondition associated with abnormal structure or expression of a gene,which method comprises a step of detecting in a biological sampleobtained from said subject polypeptide molecules comprising an aminoacid sequence in a panel of at least two amino acid sequences, whereinat least one sequence in said panel is at least 90% identical to asequence of at least 10 contiguous amino acids in a sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NO:2 and SEQID NO:10.

The present invention also includes a diagnostic system, preferably inkit form, for assaying for the presence of the polypeptide of thepresent invention in a body sample, such brain tissue, cell suspensionsor tissue sections; or a body fluid sample, such as CSF, blood, plasmaor serum, where it is desirable to detect the presence, and preferablythe amount, of the polypeptide of this invention in the sample accordingto the diagnostic methods described herein.

In a related embodiment, a nucleic acid molecule can be used as a probe(i.e., an oligonucleotide) to detect the presence of a polynucleotide ofthe present invention, a gene corresponding to a polynucleotide of thepresent invention, or a mRNA in a cell that is diagnostic for thepresence or expression of a polypeptide of the present invention in thecell. The nucleic acid molecule probes can be of a variety of lengthsfrom at least about 10, suitably about 10 to about 5000 nucleotideslong, although they will typically be about 20 to 500 nucleotides inlength. Hybridization methods are extremely well known in the art andwill not be described further here.

In a related embodiment, detection of genes corresponding to thepolynucleotides of the present invention can be conducted by primerextension reactions such as the polymerase chain reaction (PCR). To thatend, PCR primers are utilized in pairs, as is well known, based on thenucleotide sequence of the gene to be detected. Preferably, thenucleotide sequence is a portion of the nucleotide sequence of apolynucleotide of the present invention. Particularly preferred PCRprimers can be derived from any portion of a DNA sequence encoding apolypeptide of the present invention, but are preferentially fromregions which are not conserved in other cellular proteins.

Preferred PCR primer pairs useful for detecting the genes correspondingto the polynucleotides of the present invention and expression of thesegenes are described in the Examples, including the corresponding Tables.Nucleotide primers from the corresponding region of the polypeptides ofthe present invention described herein are readily prepared and used asPCR primers for detection of the presence or expression of thecorresponding gene in any of a variety of tissues.

The present invention also provides a screening assay for antiangiogeniccompounds. As disclosed herein, antiangiogenic compounds, such as TrpRSpolypeptides, can be detected by their ability to oppose the angiogeniceffects of TyrRS polypeptides on model systems such as chick allantoicmembrane (CAM), and corneal vascularization models.

The diagnostic system includes, in an amount sufficient to perform atleast one assay, a subject polypeptide of the present invention, asubject antibody or monoclonal antibody, and/or a subject nucleic acidmolecule probe of the present invention, as a separately packagedreagent.

In another embodiment, a diagnostic system, preferably in kit form, iscontemplated for assaying for the presence of the polypeptide of thepresent invention or an antibody immunoreactive with the polypeptide ofthe present invention in a body fluid sample. Such diagnostic kit wouldbe useful for monitoring the fate of a therapeutically administeredpolypeptide of the present invention or an antibody immunoreactive withthe polypeptide of the present invention. The system includes, in anamount sufficient for at least one assay, a polypeptide of the presentinvention and/or a subject antibody as a separately packagedimmunochemical reagent.

Instructions for use of the packaged reagent(s) are also typicallyincluded.

As used herein, the term “package” refers to a solid matrix or materialsuch as glass, plastic (e.g., polyethylene, polypropylene orpolycarbonate), paper, foil and the like capable of holding within fixedlimits a polypeptide, polyclonal antibody, or monoclonal antibody of thepresent invention. Thus, for example, a package can be a glass vial usedto contain milligram quantities of a contemplated polypeptide orantibody or it can be a microtiter plate well to which microgramquantities of a contemplated polypeptide or antibody have beenoperatively affixed (i.e., linked) so as to be capable of beingimmunologically bound by an antibody or antigen, respectively.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter, such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

A diagnostic system of the present invention preferably also includes alabel or indicating means capable of signaling the formation of animmunocomplex containing a polypeptide or antibody molecule of thepresent invention.

The word “complex” as used herein refers to the product of a specificbinding reaction such as an antibody-antigen or receptor-ligandreaction. Exemplary complexes are immunoreaction products.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. Any label or indicatingmeans can be linked to or incorporated in an expressed protein,polypeptide, or antibody molecule that is part of an antibody ormonoclonal antibody composition of the present invention or usedseparately, and those atoms or molecules can be used alone or inconjunction with additional reagents. Such labels are themselveswell-known in clinical diagnostic chemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelproteins methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturing them to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyanate (FITC),5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis etal., Eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference. Other suitable labeling agents areknown to those skilled in the art.

In preferred embodiments, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, or the like. In suchcases where the principal indicating group is an enzyme such as HRP orglucose oxidase, additional reagents are required to visualize the factthat a receptor-ligand complex (immunoreactant) has formed. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2′-amino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

Radioactive elements are also useful labeling agents and are usedillustratively herein. An exemplary radiolabeling agent is a radioactiveelement that produces gamma ray emissions. Elements which themselvesemit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁸I, ¹³²I and a ⁵¹Cr represent oneclass of gamma ray emission-producing radioactive element indicatinggroups. Particularly preferred is ¹²⁵I. Another group of useful labelingmeans are those elements such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N which themselvesemit positrons. The positrons so emitted produce gamma rays uponencounters with electrons present in the animal's body. Also useful is abeta emitter, such ¹¹¹indium or ³H.

The linking of labels or labeling of polypeptides and proteins is wellknown in the art. For instance, antibody molecules produced by ahybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium (see, e.g., Galfre et al., Meth. Enzymol., 73:3-46(1981)). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable (see, forexample, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23(1978); Rodwell et al., Biotech., 3:889-894 (1984); and U.S. Pat. No.4,493,795).

The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A “specific binding agent” is amolecular entity capable of selectively binding a reagent species of thepresent invention or a complex containing such a species, but is notitself a polypeptide or antibody molecule composition of the presentinvention. Exemplary specific binding agents are second antibodymolecules, complement proteins or fragments thereof, S. aureus proteinA, and the like. Preferably the specific binding agent binds the reagentspecies when that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an “ELISA”format to detect the quantity of the polypeptide of the presentinvention in a sample. “ELISA” refers to an enzyme-linked immunosorbentassay that employs an antibody or antigen bound to a solid phase and anenzyme-antigen or enzyme-antibody conjugate to detect and quantify theamount of an antigen present in a sample. A description of the ELISAtechnique is found in Sites et al., Basic and Clinical Immunology,4^(th) Ed., Chap. 22, Lange Medical Publications, Los Altos, Calif.(1982) and in U.S. Pat. No. 3,654,090; U.S. Pat. No. 3,850,752; and U.S.Pat. No. 4,016,043, which are all incorporated herein by reference.

Thus, in some embodiments, a polypeptide of the present invention, anantibody or a monoclonal antibody of the present invention can beaffixed to a solid matrix to form a solid support that comprises apackage in the subject diagnostic systems.

A reagent is typically affixed to a solid matrix by adsorption from anaqueous medium, although other modes of affixation applicable toproteins and polypeptides can be used that are well known to thoseskilled in the art. Exemplary adsorption methods are described herein.

Useful solid matrices are also well known in the art. Such materials arewater insoluble and include the cross-linked dextran available under thetrademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.),agarose; polystyrene beads of ut 1 micron (μm) to about 5 millimeters(mm) in diameter available from several suppliers (e.g., AbbottLaboratories, Chicago, Ill.), polyvinyl chloride, polystyrene,cross-linked polyacrylamide, nitrocellulose- or nylon-based webs(sheets, strips or paddles) or tubes, plates or the wells of amicrotiter plate, such as those made from polystyrene orpolyvinylchloride.

The reagent species, labeled specific binding agent, or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microtiter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnosticsystems are those customarily utilized in diagnostic systems.

The preferred embodiment of the present invention is best understood byreferring to FIGS. 1-25 and Examples 1-18. The Examples, which follow,are illustrative of specific embodiments of the invention, and varioususes thereof. They are set forth for explanatory purposes only, and arenot to be taken as limiting the invention.

Example 1 Preparation of Endotoxin-Free Recombinant TyrRS and TrpRS

Endotoxin-free recombinant human TyrRS and human TrpRS was prepared asfollows. Plasmids encoding full-length TyrRS (528 amino acid residues;FIGS. 1 and 15), truncated TyrRS (mini TyrRS, residues 1-364 offull-length TyrRS; carboxyl-domain of TyrRS, residues 359-528 offull-length TyrRS), full-length TrpRS (471 amino acid residues), ortruncated TrpRS (mini TrpRS, residues 48-471 of full-length TrpRS;supermini TrpRS, residues 71-471 of full-length TrpRS), each alsoencoding a C-terminal tag of six histidine residues, were introducedinto E. coli strain BL 21 (DE 3) (Novagen, Madison, Wis.). Human matureEMAPII, also encoding a C-terminal tag of six histidine residues, wassimilarly prepared for use in some Examples. Overexpression ofrecombinant TyrRS or TrpRS was induced by treating the cells withisopropyl β-D-thiogalactopyranoside for 4 hours. Cells were then lysedand the proteins from the supernatant purified on His•Bind® nickelaffinity columns (Novagen) according to the manufacturer's suggestedprotocol. Following purification, TrpRS proteins were incubated withphosphate-buffered saline (PBS) containing 1 μM ZnSO₄ and then free Zn²⁺was removed (Kisselev et al., 1981, Eur. J. Biochem. 120:511-17).

Endotoxin was removed from protein samples by phase separation usingTriton X-114 (Liu et al., 1997, Clin. Biochem. 30:455-63). Proteinsamples were determined to contain less than 0.01 units of endotoxin permL using an E-Toxate® gel-clot assay (Sigma, St. Louis, Mo.). Proteinconcentration was determined by the Bradford assay (Bio-Rad, Hercules,Calif.) using bovine serum albumin (BSA) as a standard.

Example 2 Cytokine Activity of Human TyrRS

Human full-length TyrRS, human mini TyrRS, human TyrRS carboxyl-terminaldomain, human EMAP II, and E. coli TyrRS were analyzed for cytokineactivity in assays examining MP or PMN chemotaxis, MP production of TNFαor tissue factor, or PMN release of myeloperoxidase.

Cells for the various cytokine assays described were prepared from acidcitrate dextrose-treated blood of normal healthy volunteers. Human PMNswere isolated from the blood by centrifugation (700×g) over Histopaque1077 and 1119 (Sigma). Fractions containing PMNs were exposed to 0.2%NaCl for 30 seconds to lyse erythrocytes, immediately restored toisotonicity by the addition of 1.6% NaCl, and then centrifuged for 10minutes. This procedure was repeated twice. Human MPs were isolated bycentrifugation on Histopaque 1077 (Sigma). The mononuclear fraction wasobtained, washed twice in Hanks' balanced salt solution, resuspended inRPMI-1640 medium (Sigma) containing 10% heat-inactivated fetal bovineserum (Sigma), plated in tissue culture flasks, and incubated in a 6%CO₂ incubator at 37° C. for 1-2 hours (Kumagai et al., 1979, J. Immunol.Methods 29:17-25). Nonadherent cells were removed by washing the flasksthree times with Hanks' balanced salt solution, and adherent cells wereharvested by incubation with calcium-magnesium free phosphate-bufferedsaline containing 2 mM EDTA for 15 minutes at 4° C., followed byextensive washing.

MP chemotaxis assays were performed in a ChemoTX microchemotaxis chamber(Neuro Probe, Gaithersburg, Md.) containing polycarbonate filters (5 μmpores) with polyvinylpyrrolidone (PVP). MPs were suspended in RPMI-1640medium containing 1% heat-inactivated fetal bovine serum, and 10⁴ cellswere added to the upper chamber. Sample proteins (1 nM) were added tothe lower compartment of chemotaxis chambers, and the chambers wereincubated for 3 hours. After incubation, nonmigrating cells wereremoved, membranes were fixed in methanol, and migrating cells werevisualized with the Hemacolor™ stain set (EM Diagnostic Systems,Gibbstown, N.J.). Migrating cells were counted in high-power fields(HPFs). Each determination shown in FIG. 2 represents the average ofnine HPF measurements.

MP TNFα production was examined following incubation of 10⁵ MPs with 1nM of sample protein for 14 hours. Aliquots of the culture supernatantwere then assayed for TNFα production using a TNFα enzyme-linkedimmunosorbent assay kit (Sigma). Each determination shown in FIG. 2represents the mean of four measurements from at least three independentexperiments.

MP tissue factor production was examined following incubation of 10⁴ MPswith 1 nM of sample protein for 4 hours. Tissue factor activity was theninferred from measurements of Factor Vlla-dependent Factor Xa formation(Wolfson et al., 1990 J. Chromatogr. 503:277-81). Each determinationshown in FIG. 2 represents the mean of four measurements from at leastthree independent experiments.

PMN release of myeloperoxidase was examined following incubation of3×10⁶ PMNs per mL with 1 nM of sample protein for 60 minutes. Thegeneration of peroxidase was then measured by the reduction of3,3′,5,5′-tetramethylbenzidine (Barker et al., 1982, FEBS Lett.150:419-23). Peroxidase activity is shown in FIG. 3 as the percent oftotal peroxidase activity where 100% peroxidase activity is defined asthe activity observed for 3×10⁶ PMNs following exposure to 10 μM phorbolester for 60 minutes. Each determination shown in FIG. 3 represents themean of four measurements from at least three independent experiments.

PMN chemotaxis assays were performed in a ChemoTX microchemotaxischamber (described herein). Sample proteins (1 nM) were added to thelower compartment of chemotaxis chambers, and 10⁴ PMNs were added to theupper compartment. Chambers were incubated for 45 min, and migratingcells were counted in HPFs. Each determination shown in FIG. 2represents the average of nine HPF measurements.

As shown in FIG. 2 (white bars), TyrRS carboxyl-terminal domain inducedMP migration to an extent comparable with that observed for EMAP II. Incontrast, no chemotaxis was observed with full-length TyrRS. The TyrRScarboxyl-terminal domain also stimulated production of TNFα (FIG. 2,gray bars) and tissue factor (FIG. 3, white bars) in MPs, induced therelease of myeloperoxidase in PMNs (FIG. 3, gray bars), and induced PMNmigration (FIG. 4). The induction of PMN migration by the TyrRScarboxyl-terminal domain and EMAP II showed the bell-shapedconcentration dependence that is characteristic of chemotactic cytokines(Wakasugi et al., supra). Full-length TyrRS had none of the propertiesobserved for the carboxyl-terminal domain (FIGS. 2-4).

The cytokine activity of the amino-terminal catalytic domain of TyrRS(mini TyrRS) examined in parallel with that of the TyrRScarboxyl-terminal domain. Mini TyrRS did not induce MP migration (FIG.2, white bars) and did not stimulate production of TNFα(FIG. 2, graybars) or tissue factor (FIG. 3, white bars) in MPs. Surprisingly, miniTyrRS did induce PMN migration (FIG. 4), and this activity showed abell-shaped concentration dependence. These results suggest that miniTyrRS is a leukocyte chemoattractant. The PMN response to mini TyrRSadded to the lower compartment of a chemotaxis chamber was attenuated bythe addition of mini TyrRS to the upper well, indicating that enhancedPMN migration was due to chemotaxis, not simply chemokinesis (stimulatedrandom movement). E. coli TyrRS, which is similar in size to human miniTyrRS, was inactive in all of the assays (FIGS. 2-4).

Example 3 Mutation of Human TyrRS ELR Motif

All α-chemokines (CXC-chemokines) that function as PMN chemoattractants,such as IL-8, have a conserved Glu-Leu-Arg (ELR) motif preceding thefirst cysteine in the amino-terminus (Becker, 1977, Arch. Pathol. Lab.Med. 101:509-13). The ELR motif is critical for receptor binding andneutrophil activation (Baggiolini et al., 1997, Annu. Rev. Immunol.15:675-705). Human mini TyrRS also possesses an ELR motif within thecatalytic domain (FIG. 5), which further comprises a Rossmannnucleotide-binding fold. The Rossman nucleotide-binding fold ischaracteristic of all class I aminoacyl-tRNA synthetases. This motif isconserved among mammalian TyrRS molecules.

The significance of the TyrRS ELR motif was examined by preparing a miniTyrRS mutant in which the ELR motif was mutated to ELQ (Glu-Leu-Gln);the Arg residue appears to be particularly important for receptorbinding (Baggiolini et al., supra). The mini TyrRS mutant was tested forcytokine activity as described in Example 2. Mini TyrRS mutant did notinduce PMN migration (FIG. 4), suggesting that, as with otherα-chemokines, the ELR motif in mini TyrRS plays an important role in PMNreceptor binding.

Example 4 Human TyrRS Binding Assay

The interaction of human mini TyrRS and PMNs was examined in bindingstudies using radioiodinated TyrRS molecules (Moser et al., 1993, J.Biol. Chem. 268:7125-28). Custom radioiodination of mini TyrRS with wasperformed by Research & Diagnostic Antibody (Richmond, Calif.). In thebinding assays, 2×10⁶ PMNs were first suspended in 120 μL of RPMI-1640medium containing 20 mM Hepes (pH 7.4) and 10 mg/mL bovine serumalbumin. The PMN cell suspension was then incubated on ice for 2 hourswith 10 nM ¹²⁵I-human mini TyrRS (specific activity of ˜60 Ci/mmol) andeither a 200-fold molar excess of unlabeled ligands or no unlabeledligands. Following incubation, cells were separated from unboundradioactivity by centrifugation at approximately 8000×g for 2 minutesthrough 500 μL of a 10% sucrose/phosphate-buffered saline (PBS) cushion.The supernatant was aspirated, and the cell sediment was resuspendedusing EcoLite (ICN Biomedicals, Irvine, Calif.) and analyzed in ascintillation counter. The maximal specific response represents 2000counts per minute. The data shown in FIG. 6 represent the mean of threeindependent measurements.

Incubation of ¹²⁵I-mini TyrRS with PMNs led to dose-dependent specificbinding at 4° C., which gave linear Scatchard plots (with an apparentdissociation constant of K_(d)=21 nM; 23,000 receptors/PMN). As shown inFIG. 6, the presence of unlabeled mini TyrRS inhibited the binding of¹²⁵I-mini TyrRS to PMNs. In contrast, human full-length TyrRS, humanmini TyrRS mutant, and E. coli TyrRS did not inhibit the binding of¹²⁵I-mini TyrRS. Thus, the lack of a PMN chemotatic effect forfull-length TyrRS, mini TyrRS mutant, and E. coli TyrRS is consistentwith their lack of PMN binding. In addition, neither the TyrRScarboxyl-terminal domain nor EMAP II inhibited ¹²⁵I-mini TyrRS bindingto PMNs. Thus, the PMN receptor for mini TyrRS differs from that for theTyrRS carboxyl-terminal domain or for mature EMAP II.

Competitive binding assays were also performed to examine the ability ofinterleukin-8 (IL-8), melanoma growth stimulatory activity (Gro), orneutrophil activating protein-2 (NAP-2) (α-chemokines possessing the ELRmotif) to bind the same PMN receptor that mini TyrRS binds. IL-8 bindsto the type A and type B IL-8 receptors (known, respectively as CXCR1and CXCR2), while Gro and NAP-2 bind to the type B IL-8 receptor(Becker, supra). In the binding assays, 2×10⁶ PMNs or basophilicleukemia cells in 120 μL of RPMI-1640 medium containing 20 mM Hepes (pH7.4) and 10 mg/mL bovine serum albumin (BSA) were incubated on ice for 2hours with 10 nM ¹²⁵I-mini TyrRS (having a specific activity of ˜60Ci/mmol) in the absence or presence of a 200-fold molar excess of eitherhuman recombinant IL-8 (Calbiochem, La Jolla, Calif.), human recombinantGro (Biosource International, Camarillo, Calif.), or human recombinantNAP-2 (Biosource International). Following incubation, cells wereseparated from unbound radioactivity as described herein.

As shown in FIG. 6, IL-8 inhibited ¹²⁵I-mini TyrRS binding almostcompletely, whereas Gro and NAP-2 did not significantly inhibit thebinding. These results suggest that mini TyrRS specifically binds to thetype A IL-8 receptor. To gain further insight into the receptor for miniTyrRS, we studied RBL2H3 rat basophilic leukemia cells that had beentransfected with the gene for IL-8 receptor type A or type B (Lee, etal., 1992, J. Biol. Chem. 267:16283-87). Untransfected RBL2H3 cellsexpress neither the type A nor type B receptors. Mini TyrRS bound withhigh affinity to cells expressing the type A IL-8 receptor (K_(d)=8 nM)as did IL-8 (K_(d)=1 nM) but not to those expressing the type B receptor(K_(d)>200 nM). Scatchard analyses demonstrated that the typeA-expressing transfectants had a similar number of binding sites forhuman mini TyrRS as for IL-8.

Example 5 Secretion of Human TyrRS From U-937 Cells

In order to examine whether human TyrRS, like human EMAP II, is secretedfrom apoptotic tumor cells, human histiocytic lymphoma U-937 cells werefirst grown in serum-free medium to induce apoptosis. Prior to growth inserum-free medium, U-937 cells were maintained in RPMI-1640 mediumcontaining 10% heat-treated FBS (Sigma), 100 U/mL penicillin and 100μg/mL streptomycin (Sigma) in an atmosphere of 6% CO₂ in air at 37° C.U-937 cells were maintained in logarithmic growth phase by routinepassage every 2-3 days. For serum-free growth, 4×10⁶ U-937 cells werecultured in RPMI-1640 medium without FBS for 24 hours. Apoptosis ofU-937 cells was verified by DNA fragment assay, in which thecharacteristic DNA ladder for apoptotic cells was observed on an agarosegel.

Cell supernatants were collected following growth of U-937 cells inserum-free media for 4, 12, or 24 hours. These supernatants were thenexamined by Western blot analysis using a rabbit polyclonal anti-TyrRSantibody. To examine proteins in the cell supernatant, 20 mL of spentculture medium was first treated with 2 mM PMSF, 10 μg/mL aprotinin, 20μg/mL leupeptin, and 10 μg/mL pepstatin A. Treated culture medium wasconcentrated using Centriprep-10 columns (Amicon, Beverly, Mass.) andthen separated on a 12.5% SDS-polyacrylamide gel. Following transferonto an Immobilon-P™ membrane (Millipore, Bedford, Mass.), blots wereblocked with PBS and 3% BSA and incubated with rabbit polyclonalanti-TyrRS antibodies. After washing, blots were incubated with a 1:4000dilution of horseradish peroxidase-linked anti-rabbit IgG (Amersham LifeScience, Arlington Heights, Ill.) for detection of TyrRS.

Cell lysates were prepared by first washing collected U-937 cells twicewith ice-cold PBS and then resuspending the cells in lysis buffercontaining 25 mM HEPES (pH 7.5), 5 mM EDTA, 5 mM dithiothreitol, 0.1%CHAPS, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/mL aprotinin, 20μg/mL leupeptin, and 10 μg/mL pepstatin A. Cells were then frozen andthawed three times in liquid nitrogen and centrifuged for 30 minutes at4° C.

Protein immunoblot analysis of the cell supernatant fraction, with apolyclonal antibody to human TyrRS, revealed that full-length TyrRS wassecreted from apoptotic tumor cells, but not from cells under normalconditions (FIG. 7A). Under apoptotic conditions, the amount of secretedhuman full-length TyrRS increased with the incubation time (FIG. 7B).After 24 hours of growth in serum-free medium, more than 50% of thetotal native TyrRS was released from the cells. A similar proportion ofmature EMAP II is secreted from U-937 cells under the same conditions(Kao, et al., 1994, J. Biol. Chem. 269:25106-19).

To exclude the possibility that the apparent secretion of TyrRS was dueto cell lysis, the activity of cytosolic lactate dehydrogenase (LDH) inthe supernatants was measured. LDH activities were determinedspectrophotometrically with a CytoTox 96 Non-Radioactive CytotoxicityAssay kit (Promega, Madison, Wis.). LDH activity in the supernatants wasless than 10% of that in cell extracts and did not increase even after72 hours of incubation. These results are consistent with the hypothesisthat the increase of TyrRS in the supernatants is due to proteinsecretion.

The permeability of the “secreting” apoptotic cells was also examinedusing Trypan Blue exclusion as a test for intact cells. The apoptoticcells did not take up the stain, indicating that cell lysis was notresponsible for the appearance of TyrRS in the apoptotic cellsupernatant. As a further control, human alanyl-tRNA synthetase (AlaRS),which possesses none of the cytokine-like motifs of human TyrRS, wasexamined for possible secretion. Protein immunoblot analysis showedthat, under the same apoptotic conditions, no AlaRS was secreted. Theactivities of four other aminoacyl-tRNA synthetases in the supernatantsor cell extracts of apoptotic U-937 cells were also studied. When cellextracts were used in assays with bovine tRNA, aminoacylation wasobserved only for alanine, isoleucine, lysine, valine, and tyrosine. Incontrast, when supernatants were used, only tyrosine was aminoacylated.

Example 6 Cleavage of Human TyrRS by PMN Elastase

In order to examine whether full-length TyrRS can be cleaved by PMNelastase, a protease released from PMNs (Wright, et al., 1992, J. Cell.Biochem. 48:344-55), full-length TyrRS was treated with PMN elastase inPBS (pH 7.4) at a protease:protein ratio of 1:3000 for 30 minutes at 37°C. Following cleavage, the sample was separated on a 12.5%SDS-polyacrylamide gel along with untreated human full-length TyrRS,human mini TyrRS, the human TyrRS carboxyl-terminal domain, and a humanextended TyrRS carboxyl-terminal domain. Immunoblot analysis wasperformed as described in Example 5.

As shown in FIG. 8A, the addition of PMN elastase to full-length TyrRSgenerated a doublet of ˜40 kD fragments and a ˜24 kD fragment. The ˜40kD fragments have a molecular weight similar to that of mini TyrRS.Sequence analysis of the ˜40 kD fragments revealed that each has anamino-terminal sequence of M-G-D-A-P (SEQ ID NO: 56), as does humanfull-length TyrRS. FIG. 8B illustrates the local sequence comparisonbetween human pro-EMAP II and human full-length TyrRS in the regionsnear their cleavage sites.

Immunoblot analysis further revealed that the ˜24 kD fragment is acarboxyl-terminal domain. A recombinant extended TyrRS carboxyl-terminaldomain (residues 344-528 of human full-length TyrRS) was prepared tomore closely reproduce the putative cleavage site recognized by PMNelastase. Both the extended TyrRS carboxyl-terminal domain and the PMNelastase ˜24 kD cleavage product migrated to similar positions on anSDS-polyacrylamide gel (FIG. 8A). Subsequent experiments demonstratedthat the extended carboxyl-terminal domain was capable of can inducingboth MP and PMN chemotaxis. In experiments examining the ability of arecombinant truncated mini TyrRS (comprising residues 1-344 of humanfull-length TyrRS in comparison to mini TyrRS which comprises TyrRSresidues 1-364) to act as a chemoattractant, the truncated mini TyrRSwas capable of functioning as a chemoattractant for PMNs but not forMPs.

In vivo cleavage analysis was performed in IL-8-stimulated PMNs, whichrelease PMN elastase (Bjørnland et al., 1998, Int. J. Oncol. 12:535-40).Recombinant human full-length TyrRS. when added to such cells, wascleaved into ˜40 kD and ˜24 kD fragments. When full-length TyrRS wasadded to nonstimulated PMNs, no TyrRS cleavage was observed. Immunoblotanalysis, using antibodies specific for the amino-terminal andcarboxyl-terminal domains indicated that the ˜40 kD and ˜24 kD fragmentscomprised mini TyrRS and the TyrRS carboxyl-terminal domain,respectively. When native TyrRS, which was isolated from apoptotic U-937cells, was added to IL-8-stimulated PMNs, the same ˜40 kD and ˜24 kDfragments were generated.

The demonstration that human full-length TyrRS can be split into twodistinct cytokines, suggests that there is a link between proteinsynthesis and signal transduction. In principle, the secretion of anessential component of the translational apparatus as an early event inapoptosis would be expected to arrest translation and thereby accelerateapoptosis. The secreted TyrRS cytokines could function as intercellularsignal transducers, attracting PMNs and thus amplifying the localconcentration of PMN elastase. This recursive cycle could enhancecleavage of secreted human TyrRS, thereby enhancing recruitment ofmacrophages to sites of apoptosis, which would promote removal of cellcorpses.

Example 7 Cytokine Activity of Non-Human TyrRS

As shown in Example 3, the ELR motif of human mini TyrRS plays animportant role in PMN receptor binding, as it does in α-chemokines Whilethe critical ELR motif of α-chemokines is conserved among mammalianTyrRS molecules, the corresponding sequence element of S. cerevisiae isNYR and that of E. coli TyrRS is ETV (FIG. 9). E. coli TyrRS neitheractivates nor binds to the IL-8 receptors on PMNs (see Example 2). Theeffect of S. cerevisiae TyrRS on PMN receptor binding and PMN migrationwas examined as described herein (see Examples 2 and 4).

As shown in Example 4, the binding of human mini TyrRS with PMNs isstrictly competitive with IL-8. This binding occurs to the IL-8 type Areceptor with a dissociation constant K_(d) of 8 nM. Incubation of¹²⁵I-mini TyrRS with PMNs led to dose-dependent specific binding at 4°C. As shown in FIG. 10, the binding of ¹²⁵I-mini TyrRS is inhibited bythe presence of excess amounts of unlabeled mini TyrRS but not by excessamounts of E. coli TyrRS. In contrast with E. coli TyrRS, S. cerevisiaeTyrRS inhibited the binding of ¹²⁵I-mini TyrRS to PMNs. The dissociationconstant for S. cerevisiae TyrRS does not appear to be more than200-fold higher than that for human mini TyrRS. These resultsdemonstrate that while a lower eukaryotic TyrRS can bind to the PMNreceptor for human mini TyrRS, a bacterial TyrRS cannot.

To determine whether the binding of S. cerevisiae TyrRS to PMNs wasassociated with cytokine activity, the chemotactic activities of humanmini TyrRS, S. cerevisiae TyrRS, and E. coli TyrRS were examined. Asshown in FIG. 11, incubation of PMNs with human mini TyrRS led to aninduction of PMN migration. In contrast, no chemotaxis was observed witheither S. cerevisiae TyrRS or E. coli TyrRS. The potential cytokineactivity of S. cerevisiae TyrRS was examined at concentrations of 0.1,1, 10, 100, and 1000 nM. No significant chemotaxis, however, wasobserved at any concentration (FIG. 11 shows the results at aconcentration of 1 nM). Thus, the binding of S. cerevisiae TyrRS to PMNsis not associated with a serendipitous cytokine function.

Because many residues and motifs are conserved among eukaryotic TyrRSmolecules that are not found in their prokaryotic counterparts, thebinding of S. cerevisiae TyrRS to PMNs could reflect its structuralsimilarity to human mini TyrRS. One reason why S. cerevisiae TyrRS doesnot have IL8-like cytokine activity might be attributed to the variation(NYR) of the sequence of critical ELR motif. As shown in Example 3, asimple change in the ELR motif of mini TyrRS inactivated its IL8-likeactivity. It is noteworthy, in this respect, that prokaryotic andeukaryote cytoplasmic TyrRS molecules cannot cross-aminoacylate theirrespective tyrosine tRNAs because of a sequence variation in a peptidemotif that is needed for discrimination of a species-specific differencein their respective tRNA sequences (Wakasugi et al., supra). Thus, bothcytokine and RNA-related activities of human TyrRS are sensitive to themost subtle sequence variations.

Example 8 Dimeric Structure for Human Mini TyrRS

Prokaryotic and lower eukaryotic TyrRS molecules form stable dimers(Quinn et al., 1995, Biochemistry 34:12489-95). The possibility that thecytokine activities of human mini TyrRS were due to its oligomericstate, and this state differed from that of prokaryotic and lowereukaryotic homologs, was examined. In order to determine the oligomericstate of mini TyrRS, the molecular weight of mini TyrRS was assayed bygel filtration chromatography on a Superose 6 HR 10/30 column (AmershamPharmacia Biotech). A gel filtration standard (Bio-Rad) that includedthyroglobulin, bovine gamma globulin, chicken ovalbumin, equinemyoglobin, and vitamin B-12 was used. Gel filtration of E. coli TyrRSand S. cerevisiae TyrRS was examined as a control. Human mini TyrRSeluted at the same position as the E. coli and S. cerevisiae TyrRS, withan estimated molecular weight of 90 kDa. This value corresponds closelyto that which would be expected for the dimeric form (84 kDa).

Under the experimental conditions of NMR or x-ray analyses, native IL-8is a dimer (Clore and Gronenborn, supra). However, at physiologicallymore relevant concentrations, monomeric and dimeric forms of IL-8 are inequilibrium, with the monomer being the prevalent form (Burrows et al.,1994, Biochemistry 33:12741-45). Dimerization-deficient IL-8 analogueswere engineered by chemical modification or by mutations of residues atthe dimer interface (Lowman et al., 1997, Protein Sci. 6:598-608). Theirstructural analyses clarified that the IL-8 monomers have the sametertiary folding as the dimer (id.). In addition, their functionalanalyses showed that the monomers have full cytokine activity in vitroand in vivo (id.). On the other hand, to mimic the dimeric form of IL-8,cross-linked single-chain dimers were designed (Leong et al., 1997,Protein Sci. 6:609-17). The results of chemotaxis and receptor bindingassays showed that the dissociation of the dimer is not required for thebiological activities (id.). Thus, dimerization of IL-8 introduces nostructural constraints for its tertiary folding or activities.Similarly, the ELR motif needed for the IL8-like activity is accessiblein the dimeric structure found here for human mini TyrRS.

Example 9 Cytokine Activity of Peptides Derived from EMAP II-LikeDomains

A synthetic peptide comprising seven residues near the amino-terminus ofhuman mature EMAP II (R-I-G-R-I-I-T; SEQ ID NO: 26) can induce PMN or MPmigration (Kao et al., 1994, J. Biol. Chem. 269:9774-82). The cytokineactivity of a synthetic peptide derived from the corresponding region ofthe TyrRS carboxyl-terminal domain (R-V-G-K-I-I-T; SEQ ID NO: 24; FIG.12) was examined in PMN and MP migration assays. The chemotactic assayswere performed as described in Example 2. Peptides used in such assayswere synthesized, purified by high performance liquid chromatography,and analyzed by mass spectroscopy by Genosys Biotechnologies, Inc.(Woodlands, Tex.). In preparing the EMAP II-derived peptide, a cysteineresidue was replaced by an arginine residue in order to enhance thestability and solubility of the synthetic peptide (FIG. 12). Thissubstitution did not alter the biological properties of the EMAP IIpeptide, as demonstrated by Kao et al., 1994, J. Biol. Chem.269:9774-82.

As shown in FIG. 13, The TyrRS carboxyl-terminal domain peptide inducedPMN migration (white bars) and MP migration (gray bars). The effect ofthe TyrRS carboxyl-terminal domain peptide on PMN and MP migrationshowed a dose-dependence similar to that of the mature EMAP II peptide.Thus, the TyrRS carboxyl-terminal domain and mature EMAP II sharesimilar peptide motifs for chemotaxis at the same positions in theirrespective sequences. Portions of S. cerevisiae Arc1p and C. elegansMetRS have high sequence homology with the TyrRS carboxyl-terminaldomain (Kleeman et al., supra; Simos et al., 1998, Mol. Cell 1:235-42).The cytokine activity of synthetic peptides corresponding to the EMAP IIR-I-G-R-I-I-T peptide and derived from S. cerevisiae Arc1p(R-V-G-F-I-Q-K; SEQ ID NO: 28) and C. elegans MetRS (R-V-G-R-I-I-K; SEQID NO: 27) were also examined in PMN and MP migration assays. As shownin FIG. 13, neither peptide induced PMN or MP migration. Thus, thecytokine activities of human mature EMAP II and the TyrRScarboxyl-terminal domain are specified by highly specific peptidesequences.

Some amino acid residues in peptides corresponding to the R-V-G-K-I-I-Tmotif from the TyrRS carboxyl-terminal domain are highly conserved amongother proteins of both prokaryotes and eukaryotes (Table I). However,heptapeptides derived from S. cerevisiae Arc1p or C. elegans MetRS didnot have EMAP II-like cytokine activities. Thus, the EMAP II-like motifin these proteins may have been originally associated with anotherbiological function. Because human EMAP II and S. cerevisiae Arc1p areknown to bind to tRNA (Simos et al., supra), the EMAP II-like motif mayhave been originally developed for RNA binding. The structure-specifictRNA binding activity of the EMAP II-like Trbp111 from Aquifex aeolicusis consistent with this possibility (Morales et al., 1999, EMBO J.18:3475-83).

These results suggest that eukaryotic TyrRS molecules had opportunitiesto gain “cytokine” functions throughout their long evolution, by theaddition of an extra domain or by accumulation of mutations. It is worthnoting that the Neurospora crassa and Podospora anserina mitochondrialTyrRS molecules also have other functions (i.e., catalysis of RNAsplicing) (Kämper et al., 1992, Mol. Cell. Biol. 12:499-511). Theseproteins contain appended domains at their amino- and carboxyl-terminiwhen compared with other mitochondrial TyrRS molecules, and theseappended domains are important for splicing (Kittle et al., 1991, GenesDev. 5:1009-21). Thus, during the molecular evolution of aminoacyl-tRNAsynthetases, attachment and removal of new domains might have occurredfrequently as these ancient enzymes acquired novel functions.

Example 10 Human Mini TyrRS Primary Structure

Human mini TyrRS differs in primary structure from more typicalα-chemokines. For example, while mini TyrRS contains an ELR motif thatis critical for receptor binding, this motif is at the middle of theRossmann fold that forms the site for synthesis of tyrosyl-adenylate. Incontrast, the ELR motif of α-chemokines is located near theamino-terminus. Also, whereas α-chemokines have conserved cysteines anda Cys-Xaa-Cys motif (where Xaa is any residue), mini TyrRS does notshare the conserved residues. Despite these differences in primarystructures, human mini TyrRS is predicted—based on the crystal structureof Bacillus stearothermophilus TyrRS (Brick et al., 1989, J. Mol. Biol.208:83-98)—to form the same six-stranded β-sheet as the α-chemokines(FIG. 14). Moreover, the predicted location of the ELR motif of humanmini TyrRS is close to that of the α-chemokines (Clore and Gronenborn,1991, J. Mol. Biol. 217:611-20).

A long amino-terminal segment precedes the ELR region of mini TyrRS. Asthe same extension occurs in the yeast and bacterial proteins examinedherein, the presence or absence of this extension cannot explain theunique cytokine activity of human mini TyrRS. Many of the ELR-containingchemokines have an amino-terminal extension that is cleaved away toactivate the chemokine (Brandt et al., 1991, Cytokine 3:311-21). Theseamino-terminal extensions may block access to the ELR motif, and it isfor that reason that activation occurs upon removal of the extensions.However, the amino-terminal region of the structural model of mini TyrRSdoes not physically block the ELR motif and therefore this region shouldbe accessible for cytokine activation. Consistent with this conclusion,in experiments using a fusion protein comprising the amino-terminus ofIL-8 and an Fab fragment, specific binding to the IL-8 receptor inducedIL-8-mediated chemotactic activity and stimulated the release ofmyeloperoxidase (Hölzer et al., 1996, Cytokine 8:214-21).

Example 11 Induction of Endothelial Cell Migration by Human TyrRS

All α-chemokines containing the ELR motif, such as IL-8, act asangiogenic factors (Strieter et al., 1995, J. Biol. Chem. 270:27348-57).In contrast, α-chemokines lacking the ELR motif act as angiostaticfactors (id.). In order to evaluate the angiogenic activity of TyrRS,which contains the ELR motif, human full-length TyrRS and human miniTyrRS were first examined for their ability to induce endothelial cellmigration. Such angiogenic assays were performed using human umbilicalvein endothelial cells (HUVECs) (Clonetics, Walkersville, Md.). Cellswere maintained in EGM-2® BulletKit® medium (Clonetics) in an atmosphereof 6% CO₂ at 37° C. according to the supplier's instructions.

Cell migration assays were performed using the modified Boyden chamber(6.5 mm Transwells) with polycarbonate membranes (8.0 μm pore size)(Costar Corp., Cambridge, Mass.) (Masood et al., 1999, Blood93:1038-44). Wells were coated overnight with 25 μg/mL human fibronectin(Biosource International, Camarillo, Calif.) in PBS and then allowed toair-dry. HUVECs were suspended in Dulbecco's modified Eagle's medium(DMEM) (Gibco-BRL, Gaithersburg, Md.) containing 0.1% BSA (Sigma) and2×10⁵ cells per well were added to the upper chamber. The chemotacticstimulus (50 nM of a given TyrRS molecule or 0.5 nM of human vascularendothelial growth factor-165 (VEGF₁₆₅) (Biosource International,Camarillo, Calif.)) was placed in the lower chamber, and the cells wereallowed to migrate for 6 hours at 37° C. in a 6% CO₂ incubator. Afterincubation, non-migrant cells were removed from the upper face of theTranswell membrane with a cotton swab and migrant cells, those attachedto the lower face, were fixed in methanol and visualized with theHemacolor® stain set (EM Diagnostic Systems, Gibbstown, N.J.). Migratingcells were counted in high-power fields (HPFs). HUVECs were suspended inmedia with the inhibitor for 30 minutes before placement in the chamber.Each determination shown in FIG. 16 represents the average of nine HPFmeasurements.

As shown in FIG. 16, human mini TyrRS stimulated induction of HUVECchemotaxis as did the positive control VEGF₁₆₅. In contrast, nochemotaxis was observed with human full-length TyrRS or human mini TyrRSmutant (containing ELQ in place of conserved ELR motif). The ability ofmini TyrRS to induce directed migration of endothelial cells supportedthe notion that mini TyrRS also may induce angiogenesis in vivo.

Example 12 Induction of In Vivo Angiogenesis by Human TyrRS

In vivo angiogenesis assays were conducted in chick chorioallantoicmembrane (CAM) (Nicolaou et al., 1998, Bioorg. Med. Chem. 6:1185-208).Ten-day-old chick embryos were purchased from Mcintyre Poultry(Lakeside, Calif.) and were incubated at 37° C. and 70% humidity. Asmall hole was made with a small crafts drill (Dremel, Emerson Electric,Racine, Wis.) directly over the air sac at the end of the egg. Theembryos were candled to determine a location to drill a second holedirectly over embryonic blood vessels. Negative pressure was applied tothe original hole, which resulted in the chorioallantoic membrane (CAM)pulling away from the shell membrane and creating a false air sac. Awindow was cut in the eggshell over the dropped CAM, exposing the CAM todirect access for experimental manipulation. Cortisone acetate-treated 5mm filter disks were soaked with a particular protein sample (25 ng ofVEGF₁₆₅ or 250 ng of a given TyrRS molecule) and the filter disks addeddirectly to the CAMs, which were relatively devoid of preexisting bloodvessels. The windows were sealed with sterile tape and incubated at 37°C. At 0, 24, and 48 hours following incubation, 3 μg of interferon-alphainducible protein (IP-10) (R & D Systems, Minneapolis, Minn.) wastopically applied to the filter disks. After 72 hours, the CAM tissueassociated with the filter disk was harvested and quantified using astereomicroscope. Angiogenesis was assessed as the number of visibleblood vessel branch points within the defined area of the filter disks.Each determination shown in FIG. 17 represents the mean from 5-8embryos.

As shown in FIG. 17, human mini TyrRS induced angiogenesis as did thepositive control human VEGF₁₆₅. Moreover, the angiogenesis stimulated byboth mini TyrRS and VEGF₁₆₅ was inhibited by the angiostaticα-chemokine, IP-10. Human mini TyrRS mutant failed to induce in vivoangiogenesis, suggesting that the ELR motif of mini TyrRS is asimportant for angiogenesis as the motif is for the angiogenic activityof α-chemokines.

Example 13 Angiostatic Effect of Human TrpRS on Cell Proliferation

Expression of mini TrpRS in human cells is highly stimulated by theaddition of interferon-γ (IFN-γ) (Shaw et al., 1999, Electrophoresis20:984-93). Expression of the α-chemokines IP-10 (interferon-γ inducibleprotein) and MIG (monokine induced by interferon-γ) has also been shownto be enhanced by IFN-γ (Kaplan et al., 1987, J. Exp. Med. 166:1098-108;Farber, 1993, Biochem. Biophys. Res. Commun. 192:223-30). Theseα-chemokines lack the ELR motif and function as angiostatic factors bothin vitro and in vivo (Strieter et al., supra). The presence in mammalianTrpRS molecules of a Rossmann nucleotide binding fold and DLT sequence,in place of the ELR motif, suggests that mammalian TrpRS molecules mayfunction as angiostatic factors.

The angiostatic activity of TrpRS was first evaluated in experimentstesting the ability of human full-length TrpRS and human mini TrpRS toinhibit human VEGF₁₆₅-induced cell proliferation. Tissue culture-treated96-well plates (Corning Costar Corp., Cambridge, Mass.) were coated with0.1% gelatin (Sigma) overnight. Cells were then seeded in thegelatinized plates at a density of 5×10³ cells per well in DMEM medium(Gibco-BRL) containing heat-inactivated fetal bovine serum (FBS) (10%,Sigma) and penicillin/streptomycin (100 units/mL-100 μg/mL, Sigma). Thefollowing day, the cells were treated with 2 μM of a given TrpRS in thepresence of 2 nM VEGF₁₆₅. After 72 hours of incubation, assays wereperformed by using the CellTiter® 96 aqueous one-solution cellproliferation assay kit (Promega, Madison, Wis.). Results of theinhibition assay are shown in FIG. 18 as the percentage of netproliferation of VEGF₁₆₅. Each determination shown represents the meanof five experiments.

As shown in FIG. 18, human full-length TrpRS exhibited no angiostaticactivity and human mini TrpRS was able to inhibit human VEGF₁₆₅-inducedcell proliferation.

Example 14 Angiostatic Effect of Human TrpRS on Endothelial CellMigration

The angiostatic activity of TrpRS was next evaluated in experimentstesting the ability of human full-length TrpRS and human mini TrpRS toinhibit human VEGF₁₆₅-induced or human mini TyrRS-induced cellmigration. Cell migration assays were performed as described in Example11 with full-length TrpRS or mini TrpRS added to VEGF₁₆₅-induced or miniTyrRS-induced HUVEC samples. HUVEC samples were treated with 0.5 nMVEGF₁₆₅, 50 nM mini TyrRS, and 500 nM of a given TrpRS. Fourmeasurements of chemotaxis were done for each protein. Eachdetermination shown in FIG. 19 represents the average of nine HPFmeasurements.

As shown in FIG. 19, human mini TrpRS inhibited human VEGF₁₆₅-inducedand human mini TyrRS-induced HUVEC chemotaxis. In contrast, humanfull-length TrpRS had no effect on VEGF₁₆₅-induced or mini TyrRS-inducedHUVEC chemotaxis.

Example 15 Angiostatic Effect of Human TrpRS on In Vivo Angiogenesis

The angiostatic activity of human full-length TrpRS and human mini TrpRSwas also analyzed in in vivo angiogenesis assays conducted in chick CAM.In vivo angiogenesis assays were performed as described in Example 12with 3 μg of full-length TrpRS or mini TrpRS added to VEGF₁₆₅-induced ormini TyrRS-induced CAM tissue.

As shown in FIG. 20, the angiogenic activity of human VEGF₁₆₅ and humanmini TyrRS was inhibited by human mini TrpRS. Human full-length TrpRShad no observable angiostatic activity.

Example 16 Secretion of Human TrpRS From U-937 Cells

As shown in Example 5, TyrRS is secreted from apoptotic tumor cellswhere it can be cleaved by PMN elastase to release mini TyrRS and anEMAP II-like carboxyl-domain. In contrast, several other tRNAsynthetases, including AlaRS, LysRS, IleRS, and ValRS, were not found tobe secreted under the same conditions. In order to determine whetherTrpRS could be secreted under these conditions, secretion assays usingU-937 cells were performed as described in Example 5. Cell supernatantswere examined by Western blot analysis using a polyclonal anti-TrpRSantibody.

As shown in FIG. 21, human full-length TrpRS was secreted from apoptoticU-937 cells, but not from U-937 cells maintained under normal (i.e.,serum) conditions. It was not possible to determine whether human miniTrpRS was also secreted from apoptotic tumor cells, since the amount ofmini TrpRS generated from full-length TrpRS is comparatively small.

Example 17 Cleavage of Human TrpRS by PMN Elastase

Cleavage of human full-length TrpRS by PMN elastase was examined usingthe procedures similar to those described in Example 6. TrpRS wastreated with PMN elastase in PBS (pH 7.4) at a protease:protein ratio of1:3000 for 0, 15, 30, or 60 minutes. Following cleavage, samples wereanalyzed on 12.5% SDS-polyacrylamide gels. As shown in FIG. 22, PMNelastase cleavage of the 54 kDa full-length TrpRS generated a majorfragment of 47 kDa and a minor fragment of 44 kDa. Both fragments weresimilar in size to the 49 kDa mini TrpRS fragment.

Western blot analysis with antibodies directed against thecarboxyl-terminal His₆-tag of the recombinant TrpRS protein revealedthat both fragments possessed the His₆-tag at their carboxyl-terminus.Thus, only the amino-terminus of two TrpRS fragments has been truncated.The amino-terminal sequences of the TrpRS fragments were determined byEdman degradation using an ABI Model 494 sequencer. Sequencing of thesefragments showed that the amino-terminal sequences were S-N-H-G-P (SEQID NO: 57) and S-A-K-G-I (SEQ ID NO: 58), indicating that theamino-terminal residues of the major and minor TrpRS fragments werelocated at positions 71 and 94, respectively, of full-length TrpRS.

The angiostatic activity of the major and minor TrpRS fragments wasanalyzed in HUVEC proliferation, HUVEC migration, and chick CAM in vivoangiogenesis assays (as described in Examples 13-15). Recombinant formsof the major and minor TrpRS fragments were prepared for use in theseassays. As shown in FIG. 23, the major TrpRS fragment (designated assupermini TrpRS) was capable of inhibiting VEGF₁₆₅-stimulated or humanmini TyrRS-stimulated angiogenesis. Supermini TrpRS was also capable ofinhibiting HUVEC proliferation and migration. Thus, the major product ofPMN elastase digestion of full-length TrpRS has angiostatic activity. Incontrast, the minor TrpRS fragment (designated as inactive TrpRS) didnot exhibit angiostatic activity.

Example 18 Human Mini TrpRS Primary Structure

Angiogenic mini TyrRS and angiostatic mini TrpRS and supermini TrpRShave their respective ELR and DLT motifs imbedded in closely similarRossmann binding fold domains. In previous experiments, α-chemokinespossessing the ELR motif have been shown to act as angiogenic factorsand α-chemokines lacking the ELR motif have been shown to act asangiostatic factors (Strieter et al., supra). It appears that thebiological balance of angiogenic (ELR) and angiostatic (non-ELR)α-chemokines regulates angiogenesis (id.). For example, theanti-tumorgenic IFN-γ induces production of IP-10 and MIG (angiostaticα-chemokines) and also attenuates the expression of IL-8 (angiogenicα-chemokine) (Gusella et al., 1993, J. Immunol. 151:2725-32). Similarly,in some cell lines TrpRS is highly expressed in the presence of IFN-γ.

Mammalian TrpRS shares some sequence similarity with semaphorin-E andneuropilin-2 (FIGS. 24 and 25). Neuropilin is a receptor for twounrelated ligands with disparate activities: VEGF₁₆₅, an angiogenicfactor, and semaphorins, which are mediators of neuronal guidance (Chenet al., 1998, Neuron 21:1273-82; Neufeld et al., 1999, FASEB J.13:9-22). Semaphorin-E is also a ligand for neuropilin-2 and semaphorinhas been shown to be capable of blocking the binding of VEGF₁₆₅ toneuropilin (Chen et al., supra). The sequence similarity of a portion ofsemaphorin-E to TrpRS suggests that human mini TrpRS and human superminiTrpRS bind neuropilin-2, thereby inhibiting VEGF₁₆₅ binding toneuropilin-2. Since the shared sequence similarity between neuropilin-2and TrpRS is located in the neuropilin-2 c-domain that is required forneuropilin-2 dimerization (Chen et al., supra) (FIG. 25), human miniTrpRS and human supermini TrpRS may inhibit the dimerization ofneuropilin-2.

Mature EMAP II has been reported to be a cytokine with anti-angiogenicproperties (Schwarz et al., 1999, J. Exp. Med. 190:341-54). Thecarboxyl-domain of human TyrRS has also been shown to have an EMAPII-like cytokine activity (see Example 2). Angiostatic mini TrpRS may beproduced by alternative splicing of the pre-mRNA or, as shown in Example17, angiostatic supermini TrpRS can be generated by PMN elastasecleav\age. PMN elastase has been shown to be present in human colorectalcarcinoma with particular enrichment at the tumor-host interface(Bjørnland et al., supra). Also, breast and non-small cell lung cancercells are known to secrete PMN elastase in vitro (Yamashita et al.,1996, Chest 109:1328-34). Thus, human TyrRS and TrpRS, which aresecreted from apoptotic cells, may be cleaved by PMN elastase at thetumor-host interface. The cleaved enzymes can then act as angiogenic andangiostatic factors that regulate tumor invasion.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

We claim:
 1. A method of suppressing angiogenesis in a mammal comprisingthe step of administering to the mammal an angiostatically effectiveamount of an isolated angiostatic truncated human tryptophanyl-tRNAsynthetase (TrpRS) polypeptide comprising a carboxyl-terminal portion ofhuman TrpRS that includes a Rossmann fold binding domain, wherein humanTrpRS has the amino acid sequence of residues 1-471 of SEQ ID NO: 10,and the polypeptide does not comprise residues 1-47 of SEQ ID NO:
 10. 2.The method of claim 1, wherein the mammal is a human.
 3. The method ofclaim 1 wherein the polypeptide comprises residues 71-471 of SEQ ID NO:10.
 4. The method of claim 1 wherein the polypeptide comprises residues48-471 of SEQ ID NO:
 10. 5. The method of claim 1 wherein thepolypeptide has a molecular weight of about 47 kD and is produced bycleavage of human TrpRS with polymorphonuclear leucocyte elastase.
 6. Amethod of treating, in a mammal, a condition that would benefit fromdecreased angiogenesis comprising the step of administering to themammal an angiostatically effective amount of an isolated angiostatictruncated human tryptophanyl-tRNA synthetase (TrpRS) polypeptide or anisolated nucleic acid comprising a polynucleotide sequence encoding thepolypeptide; the polypeptide comprising a carboxyl-terminal portion ofhuman TrpRS and including a Rossmann fold binding domain, wherein humanTrpRS has the amino acid sequence of residues 1-471 of SEQ ID NO: 10,and the polypeptide does not comprise residues 1-47 of SEQ ID NO:
 10. 7.The method of claim 6 wherein the mammal is a human.
 8. The method ofclaim 6 wherein the polypeptide comprises residues 71-471 of SEQ ID NO:10.
 9. The method of claim 6 wherein the polypeptide comprises residues48-471 of SEQ ID NO:
 10. 10. The method of claim 6 wherein thepolypeptide has a molecular weight of about 47 kD and is produced bycleavage of human TrpRS with polymorphonuclear leucocyte elastase. 11.The method of claim 6 wherein the nucleic acid comprises a leaderpolynucleotide sequence fused to the polynucleotide sequence encodingthe amino-terminal truncated human tryptophanyl-tRNA synthetasepolypeptide and in an open reading frame therewith, the leaderpolynucleotide sequence encoding a secretory peptide sequence forcontrolling transport of the polypeptide from a cell.
 12. The method ofclaim 6 wherein the condition comprises a solid tumor.
 13. The method ofclaim 12 wherein the mammal is a human.
 14. The method of claim 12wherein the polypeptide comprises residues 71-471 of SEQ ID NO:
 10. 15.The method of claim 12 wherein the polypeptide comprises residues 48-471of SEQ ID NO:
 10. 16. The method of claim 12 wherein the polypeptide hasa molecular weight of about 47 kD and is produced by cleavage of humanTrpRS with polymorphonuclear leucocyte elastase.
 17. The method of claim12 wherein the nucleic acid comprises a leader polynucleotide sequencefused to the polynucleotide sequence encoding the amino-terminaltruncated human tryptophanyl-tRNA synthetase polypeptide and in an openreading frame therewith, the leader polynucleotide sequence encoding asecretory peptide sequence for controlling transport of the polypeptidefrom a cell.
 18. The method of claim 6 wherein the condition comprises atumor metastasis.
 19. The method of claim 18 wherein the mammal is ahuman.
 20. The method of claim 18 wherein the polypeptide comprisesresidues 71-471 of SEQ ID NO:
 10. 21. The method of claim 18 wherein thepolypeptide comprises residues 48-471 of SEQ ID NO:
 10. 22. The methodof claim 18 wherein the polypeptide has a molecular weight of about 47kD and is produced by cleavage of human TrpRS with polymorphonuclearleucocyte elastase.
 23. The method of claim 18 wherein the nucleic acidcomprises a leader polynucleotide sequence fused to the polynucleotidesequence encoding the amino-terminal truncated human tryptophanyl-tRNAsynthetase polypeptide and in an open reading frame therewith, theleader polynucleotide sequence encoding a secretory peptide sequence forcontrolling transport of the polypeptide from a cell.